Method for obtaining a lipid-containing composition from microbial biomass

ABSTRACT

Methods are provided for pelletizing a microbial biomass, extracting a refined lipid composition from the pelletized biomass under supercritical conditions and distilling the refined lipid composition, at least once under short path distillation conditions, to obtain a lipid-containing fraction. Also disclosed are methods of making lipid-containing oil concentrates therefrom, by transesterifying and enriching the lipid-containing fraction.

This application claims the benefit of U.S. Provisional Application No.61/441,836, filed Feb. 11, 2011, U.S. Provisional Application No.61/441,842, filed Feb. 11, 2011, U.S. Provisional Application No.61/441,849, filed Feb. 11, 2011, U.S. Provisional Application No.61/441,854, filed Feb. 11, 2011, and U.S. Provisional Application No.61/487,019, filed May 17, 2011, which are hereby incorporated byreference in their entirety

FIELD OF THE INVENTION

The present invention relates to methods for obtaining alipid-containing fraction from microbial biomass. In particular, methodsare provided for pelletizing a microbial biomass, extracting anextracted oil from the pelletized biomass and distilling the extractedoil, at least once under short path distillation conditions, to obtain alipid-containing fraction. This lipid-containing fraction may be furtherenriched.

BACKGROUND OF THE INVENTION

Microorganisms such as filamentous fungi, yeast and algae produce avariety of lipids, including fatty acyls, glycerolipids, phospholipids,sphingolipids, saccharolipids, polyketides, sterol lipids and prenollipids. It is advantageous to extract some of these lipids from themicrobial cells in which they are produced, and thus a variety ofprocesses have been implemented.

One class of lipids commonly extracted from microbes is glycerolipids,including the fatty acid esters of glycerol (“triacylglycerols” or“TAGs”). TAGs are the primary storage unit for fatty acids, and thus maycontain long chain polyunsaturated fatty acids (“PUFAs”), as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. There has been growing interest in including PUFAs, such aseicosapentaenoic acid [“EPA”; omega-3] and docosahexaenoic acid [“DHA”;omega-3], in pharmaceutical and dietary products. Means to efficientlyand cost-effectively extract, refine and purify lipid compositionscomprising PUFAs is therefore particularly desirable.

Many typical lipid isolation procedures involve disruption of themicrobial cells (e.g., via mechanical, enzymatic or chemical means),followed by oil extraction using organic or green solvents. Thedisruption process releases the intracellular lipids from the microbialcells, which makes it readily accessible by the solvent duringextraction. After extraction, the solvent is typically removed (e.g., byevaporation, for example by application of vacuum, change of temperatureor pressure, etc.).

The resulting extracted oil is enriched in lipophilic components thataccumulate in the lipid bodies. In general, the major components of thelipid bodies consist of TAGs, ergosterol esters, other sterol esters,free ergosterol and phospholipids. PUFAs present in lipid bodies aremainly as components of TAGs, diacylglycerols, monoacylglycerols,phospholipids and free fatty acids. The extracted oil may then besubsequently refined, to produce a highly purified TAG fraction enrichedin PUFAs. Final specifications concerning the purified TAG fraction maybe application-dependent, for example, depending on whether the oil isto be used as an additive or supplement (e.g., in food compositions,infant formulas, animal feeds, etc.), in cosmetic or pharmaceuticalcompositions, etc. Acceptable contaminant standards are eitherself-imposed (wherein a particular contaminant results in an undesirableproperty, e.g., haziness/cloudiness, odor) or determined by externalnutrition councils (e.g., A Voluntary Monograph Of The Council forResponsible Nutrition [Washington, D.C.], March 2006, specifies themaximum acid, peroxide, anisidine, TOTOX, polychlorinateddibenzo-para-dioxin and polychlorinated dibenzofuran values for omega-3EPA, omega-3 DHA and mixtures thereof).

U.S. Pat. No. 6,727,373 discloses a microbial PUFA-containing oil with ahigh triglyceride content and a high oxidative stability. In addition, amethod is described for the recovery of such oil from a microbialbiomass derived from a pasteurized fermentation broth, wherein themicrobial biomass is subjected to extrusion to form granular particles,dried, and the oil is then extracted from the dried granules using anappropriate solvent.

U.S. Pat. No. 6,258,964 discloses a method of extracting liposolublecomponents contained in microbial cells, wherein the method requiresdrying microbial cells containing liposoluble components, simultaneouslydisrupting and molding the dried microbial cells into pellets by use ofan extruder, and extracting the contained liposoluble components by useof an organic solvent.

U.S. Pat. Appl. Pub. No. 2009/0227678 discloses a process for obtaininglipid from a composition comprising cells and water, the processcomprising contacting the composition with a desiccant, and recoveringthe lipid from the cells.

U.S. Pat. No. 4,675,132 discloses a process for the concentration ofPUFA moieties in a fish oil containing relatively low proportions ofsaturated and monounsaturated fatty acid moieties of the same chainlength as the PUFA moieties to be concentrated, which comprisestransesterifying fish oil glycerides with a lower alkanol to form amixture of lower alkyl fatty acid esters, and extracting said esterswith carbon dioxide (CO₂) under supercritical conditions.

A process flow diagram developed for a continuous countercurrentsupercritical CO₂ fractionation process that produces high concentrationEPA is disclosed by V. J. Krukonis et al. (Adv. Seafood Biochem., Pap.Am. Chem. Soc. Annu. Meet. (1992), Meeting Date 1987, 169-179). Thefeedstock for the process is urea-crystallized ethyl esters of menhadenoil, and the basis for the design is a product concentration of 90% EPA(ethyl ester) at a yield of 90%.

Methods in which the distribution of TAGs, diacylglycerols,monoacylglycerols, and free fatty acids can be adjusted in aPUFA-containing lipid composition are sought. Methods for obtainingPUFA-containing lipid compositions which have improved oxidativestability are desired. Methods for obtaining PUFA-containing lipidcompositions enriched in TAGs are also desired, as are economicalmethods for obtaining such compositions.

U.S. Pat. No. 6,166,230 (GIST-Brocades) describes a process for treatinga microbial oil comprising PUFAs (e.g., from Mortierella alpina) with apolar solvent to extract at least one sterol (e.g., desmosterol) that issoluble in the solvent and then separating at least some of the solventcontaining the sterol from the oil, wherein the oil has a sterol contentof less than 1.5%.

U.S. Pat. No. 7,695,626 (Martek) describes a process for recoveringneutral lipids comprising PUFAs from a microbial biomass (e.g.,Schizochytrium), said process comprising the steps of contacting thebiomass with a nonpolar solvent to recover lipid in an extractionprocess, refining and/or bleaching and/or deodorizing the lipidcomposition, adding a polar solvent to the lipid composition, coolingthe mixture to selectively precipitate at least one other compound(e.g., trisaturated glycerides, phosphorus-containing materials, waxesters, saturated fatty acid containing sterol esters, sterols,squalene, hydrocarbons) and then removing this undesirable compound fromthe lipid composition.

Previous methods have not utilized techniques of short path distillationas an effective means to avoid exposing PUFAs, specifically highlyunsaturated fatty acids, to high temperatures and remove ergosterol(ergosta-5,7,22-trien-3β-ol; CAS Registry Number 57-87-4) contaminantsfrom microbial oils.

Int'l. Appl. Pub. No. WO 2011/080503 A2 discloses a chromatographicseparation process for recovering a PUFA product, from a feed mixture,comprising introducing the feed mixture to a simulated or actual movingbed chromatography apparatus having a plurality of linked chromatographycolumns containing, as eluent, an aqueous alcohol, wherein the apparatushas a plurality of zones comprising at least a first zone and secondzone, each zone having an extract stream and a raffinate stream fromwhich liquid can be collected from said plurality of linkedchromatography columns, and wherein (a) a raffinate stream containingthe PUFA product together with more polar components is collected from acolumn in the first zone and introduced to a nonadjacent column in thesecond zone, and/or (b) an extract stream containing the PUFA producttogether with less polar components is collected from a column in thesecond zone and introduced to a nonadjacent column in the first zone,said PUFA product being separated from different components of the feedmixture in each zone. Various fish oil derived feedstocks were purifiedto produce 85 to greater than 98% EPA ethyl esters. Although Int'l.Appl. Pub. No. WO 2001/080503 A2 demonstrated processes to recover EPAand DHA in high purity from fish oils, the disclosure also states thatsuitable feed mixtures for fractionating may be obtained from “syntheticsources including oils obtained from genetically modified plants,animals and microorganisms including yeasts”. Further, “geneticallymodified yeast is particularly suitable when the desired PUFA product isEPA”.

SUMMARY OF THE INVENTION

In a first embodiment, the invention concerns a method comprising:

-   -   (a) pelletizing a microbial biomass having a moisture level and        comprising oil-containing microbes;    -   (b) extracting the pelletized microbial biomass of step (a) to        produce an extracted oil; and,    -   (c) distilling the extracted oil of step (b) at least once under        short path distillation conditions, wherein said distillation        produces a distillate fraction and a lipid-containing fraction.

In a second embodiment of the method, the oil-containing microbes areselected from the group consisting of yeast, algae, fungi, bacteria,euglenoids, stramenopiles and oomycetes. Preferably, the yeast isYarrowia.

In a third embodiment of the method, the oil-containing microbescomprise at least one polyunsaturated fatty acid in the oil, wherein thepolyunsaturated fatty acids are preferably selected from the groupconsisting of: linoleic acid, gamma-linolenic acid, eicosadienoic acid,dihomo-gamma-linolenic acid, arachidonic acid, docosatetraenoic acid,omega-6 docosapentaenoic acid, alpha-linolenic acid, stearidonic acid,eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,omega-3 docosapentaenoic acid and docosahexaenoic acid.

In a fourth embodiment of the method, the moisture level of themicrobial biomass is in the range of about 1 to 10 weight percent.

In a fifth embodiment of the method, said step (a) pelletizing themicrobial biomass comprises:

-   -   (1) mixing the microbial biomass and at least one grinding agent        capable of absorbing oil to provide a disrupted biomass mix        comprising disrupted microbial biomass;    -   (2) blending the disrupted biomass mix with at least one binding        agent to provide a fixable mix capable of forming a solid        pellet; and,    -   (3) forming said fixable mix into solid pellets to provide a        pelletized microbial biomass.        Preferably, steps (1) mixing the microbial biomass and (2)        blending at least one binding agent are performed in an        extruder, are performed simultaneously, or are performed        simultaneously in an extruder; and, step (3) forming said solid        pellet from said fixable mix comprises a step selected from the        group consisting of:    -   (i) extruding said fixable mix through a die to form strands;    -   (ii) drying and breaking said strands; and,    -   (iii) combinations of step (i) extruding said fixable mix        through a die to form strands and step (ii) drying and breaking        said strands.

In a sixth embodiment of the present method, the disrupted microbialbiomass is produced in a twin screw extruder comprising: (a) a totalspecific energy input of about 0.04 to 0.4 KW/(kg/hr); (b) a compactionzone using bushing elements with progressively shorter pitch length;and, (c) a compression zone using flow restriction; wherein thecompaction zone is prior to the compression zone within the extruder.

In a seventh embodiment of the method, the at least one grinding agentpreferably has a property selected from the group consisting of:

-   -   (a) said at least one grinding agent is a particle having a Moh        hardness of 2.0 to 6.0 and an oil absorption coefficient of 0.8        or higher as determined according to ASTM Method D1483-60;    -   (b) said at least one grinding agent is selected from the group        consisting of silica and silicate; and,    -   (c) said at least one grinding agent is present at about 1 to 20        weight percent, based on the summation of the weights of        microbial biomass, grinding agent and binding agent in the solid        pellet.        The at least one binding agent preferably has a property        selected from the group consisting of:    -   (a) said at least one binding agent is selected from water and        carbohydrates selected from the group consisting of sucrose,        lactose, fructose, glucose, and soluble starch; and,    -   (b) said at least one binding agent is present at about 0.5 to        10 weight percent, based on the summation of the weights of        microbial biomass, grinding agent and binding agent in the solid        pellet.

In an eighth embodiment of the method, the pellets have a propertyselected from the group consisting of:

-   -   (a) said pellets have an average diameter of about 0.5 to about        1.5 mm and an average length of about 2.0 to about 8.0 mm; and,    -   (b) said pellets comprise about 70 to about 98.5 weight percent        of microbial biomass comprising oil-containing microbes, about 1        to about 20 weight percent of at least one grinding agent        capable of absorbing oil and about 0.5 to 10 weight percent of        at least one binding agent, based on the summation of the        weights of microbial biomass, grinding agent and binding agent        in the solid pellet.

In a ninth embodiment of the method, the extracting is performed with anorganic solvent to produce an extracted oil and said extracted oil isdegummed and optionally bleached prior to said step (c) distilling theextracted oil.

In a tenth embodiment of the method, the extracting comprises:

-   -   (1) processing the pelletized microbial biomass with a solvent        comprising liquid or supercritical fluid carbon dioxide, wherein        said pelletized microbial biomass comprising oil-containing        microbes further comprises at least one polyunsaturated fatty        acid in the oil, to obtain:        -   (i) an extract comprising a lipid fraction substantially            free of phospholipids; and,        -   (ii) a residual biomass comprising phospholipids; and,    -   (2) fractionating the extract obtained in step (1), part (i) at        least once to obtain an extracted oil having a refined lipid        composition comprising at least one polyunsaturated fatty acid,        wherein the refined lipid composition is enriched in        triacylglycerols relative to the oil composition of pelletized        microbial biomass that is not processed with a solvent.

In an eleventh embodiment of the method, the extracted oil of step (b)comprises a sterol fraction, the distillate fraction of step (c)comprises the sterol and the lipid-containing fraction of step (c)comprises a reduced amount of the sterol when compared to the amount ofthe sterol in the extracted oil that has not been subjected to shortpath distillation. The sterol fraction may comprise one or more sterolsselected from the group consisting of: stigmasterol, ergosterol,brassicasterol, campesterol, β-sitosterol and desmosterol.

In a twelfth embodiment of the method, the extracted oil having arefined lipid composition comprising at least one polyunsaturated fattyacid and enriched in triacylglycerols relative to the oil composition ofpelletized microbial biomass that is not processed with a solventfurther comprises a sterol fraction of at least 300 mg/100 g. Upondistillation at least once under short path distillation conditions, adistillate fraction is produced comprising the sterol and alipid-containing fraction is produced comprising triacylglycerols and areduced amount of sterol when compared to the amount of sterol in theextracted oil having a refined lipid composition that has not beensubjected to short path distillation.

In a thirteenth embodiment, the method further comprises:

-   -   (d) transesterifying the lipid-containing fraction of step (c);        and,    -   (e) enriching the transesterified lipid-containing fraction of        step (d) to obtain an oil concentrate.

In a fourteenth embodiment, the oil-containing microbes accumulate inexcess of 25% of their dry cell weight as microbial oil; and, themicrobial oil comprises 30 to 70 weight percent of eicosapentaenoicacid, measured as a weight percent of total fatty acids, and issubstantially free of docosahexaenoic acid; and, the enriching of step(e) is by a combination of at least two processes, said first processcomprising fractional distillation and said second process selected fromthe group consisting of: urea adduct formation, liquid chromatography,supercritical fluid chromatography, simulated moving bed chromatography,actual moving bed chromatography and combinations thereof; and, the oilconcentrate is an eicosapentaenoic acid concentrate comprising at least70 weight percent of eicosapentaenoic acid, measured as a weight percentof oil, and substantially free of docosahexaenoic acid.

BIOLOGICAL DEPOSITS

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, accession numbersand dates of deposit.

Biological Material Accession No. Date of Deposit Yarrowia lipolyticaY4128 ATCC PTA-8614 Aug. 23, 2007 Yarrowia lipolytica Y8412 ATCCPTA-10026 May 14, 2009 Yarrowia lipolytica Y8259 ATCC PTA-10027 May 14,2009

The biological materials listed above were deposited under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. The listed depositwill be maintained in the indicated international depository for atleast 30 years and will be made available to the public upon the grantof a patent disclosing it. The availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by government action.

Yarrowia lipolytica Y4305 was derived from Y. lipolytica Y4128,according to the methodology described in U.S. Pat. Appl. Pub. No.2009-0093543-A1. Y. lipolytica Y9502 was derived from Yarrowialipolytica Y8412, according to the methodology described in U.S. Pat.Appl. Pub. No. 2010-0317072-A1. Similarly, Yarrowia lipolytica Y8672 wasderived from Y. lipolytica Y8259, according to the methodology describedin U.S. Pat. Appl. Pub. No. 2010-0317072-A1.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

FIG. 1 illustrates a custom high-pressure extraction apparatusflowsheet.

FIG. 2 schematically illustrates one embodiment of extraction in whichpelletized microbial biomass is contacted with CO₂ to obtain an extractwhich is then fractionated.

FIG. 3 schematically illustrates one embodiment of extraction, in whichmicrobial biomass is contacted with CO₂ to obtain an extract.

FIG. 4 is a graphical representation of the extraction curve obtained inExample 12.

FIG. 5 provides an overview of the processes of the invention, in theform of a flowchart. Specifically, a microbial fermentation producesuntreated microbial biomass, which is then pelletized. Oil extraction ofthe solid pellets results in residual biomass and extracted oil.Distillation of the extracted oil using short path distillation (SPD)conditions produces a distillate fraction and a lipid-containingfraction, which may optionally be further transesterified and enrichedto yield an oil concentrate.

FIG. 6 provides plasmid maps for the following: (A) pZKUM; and, (B)pZKL3-9DP9N.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID Nos:1-8 are open reading frames encoding genes, proteins (orportions thereof), or plasmids, as identified in Table 1.

TABLE 1 Summary Of Nucleic Acid And Protein SEQ ID Numbers Nucleic acidProtein Description SEQ ID NO. SEQ ID NO. Plasmid pZKUM 1 — (4313 bp)Plasmid pZKL3-9DP9N 2 — (13565 bp)  Synthetic mutant delta-9 elongase,derived 3 4 from Euglena gracilis (“EgD9Es-L35G”)  (777 bp) (258 AA)Yarrowia lipolytica delta-9 desaturase gene 5 6 (GenBank Accession No.XM_501496) (1449 bp) (482 AA) Yarrowia lipolytica choline-phosphate 7 8cytidylyl-transferase gene (GenBank (1101 bp) (366 AA) Accession No.XM_502978)

DETAILED DESCRIPTION OF THE INVENTION

The disclosures of all patent and non-patent literature cited herein arehereby incorporated by reference in their entireties.

When an amount, concentration, or other value or parameter is given aseither a range, preferred range, or a list of upper preferable valuesand lower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limit orpreferred value and any lower range limit or preferred value, regardlessof whether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope of the invention be limitedto the specific values recited when defining a range.

As used herein, the terms “comprises”, “comprising”, “includes”,“including”, “has”, “having”, “contains” or “containing”, or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, mixture, process, method, article, or apparatusthat comprises a list of elements is not necessarily limited to onlythose elements but may include other elements not expressly listed orinherent to such composition, mixture, process, method, article, orapparatus. Further, unless expressly stated to the contrary, “or” refersto an inclusive or and not to an exclusive or. For example, a conditionA or B is satisfied by any one of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e., occurrences) of the element or component.Therefore, “a” or “an” should be read to include one or at least one,and the singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

As used herein the term “invention” or “present invention” is intendedto refer to all aspects and embodiments of the invention as described inthe claims and specification herein and should not be read so as to belimited to any particular embodiment or aspect.

The following definitions are used in this disclosure:

“Carbon dioxide” is abbreviated as “CO₂”.

“American Type Culture Collection” is abbreviated as “ATCC”.

“Polyunsaturated fatty acid(s)” is abbreviated as “PUFA(s)”.

“Phospholipids” are abbreviated as “PLs”.

“Triacylglycerols” are abbreviated as “TAGs”.

“Free fatty acids” are abbreviated as “FFAs”.

“Total fatty acids” are abbreviated as “TFAs”.

“Fatty acid methyl esters” are abbreviated as “FAMEs”.

“Ethyl esters” are abbreviated as “EEs”.

“Dry cell weight” is abbreviated as “DCW”.

“Millitorr” is abbreviated as “mTorr”.

“Short path distillation” is abbreviated as “SPD”.

As used herein the term “microbial biomass” refers to microbial cellularmaterial from a microbial fermentation of oil-containing microbes,conducted to produce microbial oil. The microbial biomass may be in theform of whole cells, whole cell lysates, homogenized cells, partiallyhydrolyzed cellular material, and/or disrupted cells. Preferably, themicrobial oil comprises at least one PUFA.

The term “untreated microbial biomass” refers to microbial biomass priorto extraction with a solvent. Optionally, untreated microbial biomassmay be subjected to at least one mechanical process (e.g., by drying thebiomass, disrupting the biomass, pelletizing the biomass, or acombination of these) prior to extraction with a solvent. The terms“untreated microbial biomass” and “unrefined microbial biomass” are usedinterchangeably herein.

The term “pelletizing” or “pelletization” refers to a process forproducing a solid pellet.

The term “solid pellet” refers to a pellet having structural rigidityand resistance to changes of shape or volume. Solid pellets desirablyare non-tacky at room temperature. A large plurality of the solidpellets may be packed together for many days without degradation of thepellet structure, and without binding together; thus, the largeplurality desirably is a free-flowing pelletized composition. Solidpellets are formed herein from microbial biomass via a process of“pelletization” and thus may also be referred to as “pelletizedmicrobial biomass”.

The term “disrupted biomass mix” refers to the product obtained bymixing microbial biomass and at least one grinding agent. The disruptedbiomass mix comprises disrupted microbial biomass.

The term “disrupted microbial biomass” refers to microbial biomass thathas been subjected to a process of disruption, wherein said disruptionresults in a disruption efficiency of at least 50% of the microbialbiomass.

The term “disruption efficiency” refers to the percent of cells wallsthat have been fractured or ruptured during processing, as determinedqualitatively by optical visualization or as determined quantitativelyaccording to the following formula: % disruption efficiency=(% freeoil*100) divided by (% total oil), wherein % free oil and % total oilare measured for the solid pellet. Increased disruption efficiency ofthe microbial biomass typically leads to increased extraction yields ofthe microbial oil contained within the microbial biomass.

The term “percent total oil” refers to the total amount of all oil(e.g., including fatty acids from neutral lipid fractions [DAGs, MAGs,TAGs], free fatty acids, phospholipids, etc. present within cellularmembranes, lipid bodies, etc.) that is present within a solid pelletsample. Percent total oil is effectively measured by converting allfatty acids within a pelletized sample that has been subjected tomechanical disruption, followed by methanolysis and methylation of acyllipids. Thus, the sum of the fatty acids (expressed in triglycerideform) is taken to be the total oil content of the sample. In the presentinvention, percent total oil is preferentially determined by gentlygrinding a solid pellet into a fine powder using a mortar and pestle,and then weighing aliquots (in triplicate) for analysis. The fatty acidsin the sample (existing primarily as triglycerides) are converted to thecorresponding methyl esters by reaction with acetyl chloride/methanol at80° C. A C15:0 internal standard is then added in known amounts to eachsample for calibration purposes. Determination of the individual fattyacids is made by capillary gas chromatography with flame ionizationdetection (GC/FID). And, the sum of the fatty acids (expressed intriglyceride form) is taken to be the total oil content of the sample.

The term “percent free oil” refers to the amount of free and unbound oil(e.g., fatty acids expressed in triglyceride form, but not allphospholipids) that is readily available for extraction from aparticular solid pellet sample. Thus, for example, an analysis ofpercent free oil will not include oil that is present in non-disruptedmembrane-bound lipid bodies. In the present invention, percent free oilis preferentially determined by stirring a sample with n-heptane,centrifuging, and then evaporating the supernatant to dryness. Theresulting residual oil is then determined gravimetrically and expressedas a weight percentage of the original sample.

The term “grinding agent” refers to an agent, capable of absorbing oilthat is mixed with microbial biomass to yield disrupted biomass mix.Preferably, the at least one grinding agent is present at about 1 to 50parts, based on 100 parts of microbial biomass. In some preferredembodiments, the grinding agent is a silica or silicate. Other preferredproperties of the grinding agent are discussed infra.

The term “fixable mix” refers to the product obtained by blending atleast one binding agent with disrupted biomass mix. The fixable mix is amixture capable of forming a solid pellet upon removal of solvent (e.g.,removal of water in a drying step).

The term “binding agent” refers to an agent that is blended withdisrupted biomass mix to yield a fixable mix. Preferably, the at leastone binding agent is present at about 0.5 to 20 parts, based on 100parts of microbial biomass. In some preferred embodiments, the bindingagent is a carbohydrate. Other preferred properties of the binding agentare discussed infra.

As used herein the term “residual biomass” refers to microbial cellularmaterial from a microbial fermentation that is conducted to producemicrobial oil, which has been extracted at least once with a solvent(e.g., an inorganic or organic solvent). When the initial microbialbiomass subjected to extraction is in the form of a solid pellet, theresidual biomass may be referred to as a “residual pellet”.

The term “reduced” or “depleted” means having a smaller quantity, forexample a quantity only slightly less than the original quantity, or forexample a quantity completely lacking in the specified material, andincluding all quantities in between.

The term “lipids” refer to any fat-soluble (i.e., lipophilic),naturally-occurring molecule. Lipids are a diverse group of compoundsthat have many key biological functions, such as structural componentsof cell membranes, energy storage sources and intermediates in signalingpathways. Lipids may be broadly defined as hydrophobic or amphiphilicsmall molecules that originate entirely or in part from either ketoacylor isoprene groups. A general overview of lipids, based on the LipidMetabolites and Pathways Strategy (LIPID MAPS) classification system(National Institute of General Medical Sciences, Bethesda, Md.), isshown below in Table 2.

TABLE 2 Overview Of Lipid Classes Structural Building Block LipidCategory Examples Of Lipid Classes Derived Fatty Acyls Includes fattyacids, eicosanoids, fatty from esters and fatty amides condensationGlycerolipids Includes mainly mono-, di- and tri- of ketoacylsubstituted glycerols, the most well-known subunits being the fatty acidesters of glycerol (triacylglycerols) Glycero- Includesphosphatidylcholine, phospholipids phosphatidylethanolamine, phospha- ortidylserine, phosphatidylinositols and Phospholipids phosphatidic acidsSphingolipids Includes ceramides, phospho-sphingolipids (e.g.,sphingomyelins), glycosphingolipids (e.g., gangliosides), sphingosine,cerebrosides Saccharolipids Includes acylaminosugars, acylamino- sugarglycans, acyltrehaloses, acyltrehalose glycans Polyketides Includeshalogenated acetogenins, polyenes, linear tetracyclines, polyetherantibiotics, flavonoids, aromatic polyketides Derived Sterol LipidsIncludes sterols (e.g., cholesterol), C18 from steroids (e.g.,estrogens), C19 steroids condensation (e.g., androgens), C21 steroids(e.g., of isoprene progestagens, glucocorticoids and mineral- subunitsocorticoids), secosteroids, bile acids Prenol Lipids Includesisoprenoids, carotenoids, quinones, hydroquinones, polyprenols,hopanoids

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. In oleaginous organisms, oil constitutes amajor part of the total lipid. “Oil” is composed primarily oftriacylglycerols (TAGs) but may also contain other neutral lipids,phospholipids (PLs) and free fatty acids (FFAs). The fatty acidcomposition in the oil and the fatty acid composition of the total lipidare generally similar; thus, an increase or decrease in theconcentration of PUFAs in the total lipid will correspond with anincrease or decrease in the concentration of PUFAs in the oil, and viceversa.

A “microbial oil” is an oil produced by a microbe. This generic term mayrefer to a non-concentrated microbial oil, an extracted oil, alipid-containing fraction, a purified oil or a concentrated microbialoil, as further defined hereinbelow. After purification or enrichment ofa specific fatty acid in a microbial oil, the oil can exist in variouschemical forms (e.g., in the form of triacylglycerols, alkyl esters,salts or free fatty acids).

The term “extracted oil” or “crude oil” (as the terms can be usedinterchangeably herein) refers to an oil that has been separated fromcellular materials, such as the microorganism in which the oil wassynthesized. Extracted oils are obtained through a wide variety ofmethods, the simplest of which involves physical means alone. Forexample, mechanical crushing using various press configurations (e.g.,screw, expeller, piston, bead beaters, etc.) can separate oil fromcellular materials. Alternatively, oil extraction can occur viatreatment with various organic solvents (e.g., hexane, iso-hexane),enzymatic extraction, osmotic shock, ultrasonic extraction,supercritical fluid extraction (e.g., CO₂ extraction), saponificationand combinations of these methods. Often, the amount of oil that may beextracted from the microorganism is proportional to the disruptionefficiency. Further purification or concentration of an extracted oil isoptional.

The term “non-concentrated microbial oil” means that the extracted oilhas not been substantially enriched in one or more fatty acids. Thus,the fatty acid composition of the “non-concentrated microbial oil” issubstantially similar to the fatty acid composition of the microbial oilas produced by the microorganism. The non-concentrated microbial oil maybe non-concentrated extracted oil or non-concentrated purified oil.

The term “extracted oil having a refined lipid composition” or “refinedlipid composition” refers to a microbial oil composition that is theproduct of a supercritical carbon dioxide (CO₂) extraction as disclosedin U.S. Pat. Pub. No. 2011-0263709-A1. Thus, the refined lipidcomposition is an extracted oil. The refined lipid composition maycomprise neutral lipids and/or FFAs while being substantially free ofPLs. The refined lipid composition preferably has less than 30 ppmphosphorous, and more preferably less than 20 ppm phosphorous, asdetermined by the American Oil Chemists' Society (AOCS) Official MethodCa 20-99 entitled “Analysis for Phosphorus in Oil by Inductively CoupledPlasma Optical Emission Spectroscopy” (Official Methods and RecommendedPractices of the AOCS, 6^(th) ed., Urbana, Ill., AOCS, 2009,incorporated herein by reference). The refined lipid composition may beenriched in TAGs relative to the oil composition of the microbialbiomass and may optionally comprise a sterol fraction. The refined lipidcomposition may undergo further purification, such as via short pathdistillation as described herein, to produce a “purified oil” or“lipid-containing fraction”.

The term “degumming” refers to a process that reduces the concentrationof phospholipids and other impurities from an extracted oil.

The term “bleaching” refers to a process that reduces the concentrationof pigments/color compounds and residual metals from an extracted oil.

The term “sterols” or “sterol fraction” refers to biological componentsthat affect membrane permeability within cells. Sterols have beenisolated from all major groups of living organisms, although there isdiversity in the predominant sterol isolated. The predominant sterol inhigher animals is cholesterol, while β-sitosterol is commonly thepredominant sterol in higher plants (although it is frequentlyaccompanied by campesterol and stigmasterol). Generalization concerningthe predominant sterol(s) found in microbes is more difficult, as thecomposition depends on the particular microbial species. For example,the oleaginous yeast Yarrowia lipolytica predominantly comprisesergosterol, fungi of the genus Morteriella predominantly comprisecholesterol and desmosterol, and stramenopiles of the genusSchizochytrium predominantly comprise brassicasterol and stigmasterol. Asummary of sterols often found in sterol-containing microbial oils isshown below in Table 3; in contrast, these sterols are not typicallyfound in fish oils. When present in microbial oils, the sterols of Table3 tend to precipitate out of the extracted oil due to high meltingpoints and reduced solubility at lower storage temperatures, whichresult in a cloudy oil. It is highly desirable to minimize undesirablecloudiness in the extracted oil or oil products therefrom by reducingthe concentration of these sterols.

TABLE 3 Sterols In Sterol-Containing Microbial Oils Common Name ChemicalName CAS Registry No. Stigmasterol Stigmasta-5,22-dien-3-ol  83-48-7Ergosterol Ergosta-5,7,22-trien-3β-ol 474-67-9 BrassicasterolErgosta-5,22-dien-3β-ol  57-87-4 Campesterol (24R)-Ergost-5-en-3β-ol474-62-4 β-Sitosterol Stiqmast-5-en-3-ol,  83-46-5 DesmosterolCholesta-5,24-dien-3β-ol 313-04-2

The term “distilling” refers to a method of separating mixtures based ondifferences in their volatilities in a boiling liquid mixture.Distillation is a unit operation, or a physical separation process, andnot a chemical reaction.

The term “short path distillation” (“SPD”) refers to a separation methodoperating under an extremely high vacuum, in which the SPD device isequipped with an internal condenser in close proximity to theevaporator, such that volatile compounds from the material to bedistilled after evaporation travel only a short distance to thecondensing surface. As a result, there is minimal thermal degradationfrom this separation method.

The term “purified oil” refers to an extracted oil having reducedconcentrations of impurities, such as phospholipids, trace metals, freefatty acids, color compounds, minor oxidation products, volatile and/orodorous compounds, and sterols (e.g., ergosterol, brassicasterol,stigmasterol, cholesterol), as compared to the concentrations ofimpurities in the extracted oil. Purification processes do not typicallyconcentrate or enrich the microbial oil, such that a particular fattyacid(s) is substantially enriched, and thus purified oil is most oftennon-concentrated.

The terms “lipid-containing fraction” and “SPD-purified oil” are usedinterchangeably herein. These terms refer to an extracted microbial oilcontaining a TAG-fraction comprising one or more PUFAs, said oil havingundergone a process of distillation at least once under SPD conditions.If a sterol fraction is present in the extracted oil, the distillationprocess reduces the amount of sterol in the lipid-containing fraction,as compared to the sterol content in the oil prior to short pathdistillation.

Although SPD can concentrate ethyl esters, methyl esters and free fattyacids, the process does not typically concentrate TAGs (e.g., unlessoperated at extremely high temperatures which then leads todecomposition of TAGs). Since the majority of PUFAs in thelipid-containing fraction are in the form of TAGs, and the SPD processdoes not typically concentrate TAGs such that a particular fatty acid(s)is substantially enriched, the lipid-containing fraction is consideredto be non-concentrated most often for the purposes described herein.

The term “transesterification” refers to a chemical reaction, catalyzedby an acid or base catalyst, in which an ester of a fatty acid isconverted to a different ester of the fatty acid.

“Fatty acid ethyl esters” [“FAEEs”] refer to a chemical form of lipidsthat are generally synthetically derived by reacting free fatty acids ortheir derivatives with ethanol, in a process of esterification ortransesterification.

The term “enrichment” refers to a process to increase the concentrationof one or more fatty acids in a microbial oil, relative to theconcentration of the one or more fatty acids in the non-concentratedmicrobial oil. For example, as discussed herein, a microbial oilcomprising 30 to 70 wt % of a desired PUFA, measured as a wt % of TFAs,is enriched or concentrated to produce an “oil concentrate”.

The term “oil concentrate” refers to an oil comprising at least 70 wt %of a desired PUFA, measured as a wt % of oil. Preferably, the oilconcentrate is obtained from a microbial oil comprising 30 to 70 wt % ofthe desired PUFA, measured as a wt % of total fatty acids, wherein saidmicrobial oil is obtained from an oil-containing microbe thataccumulates in excess of 25% of its dry cell weight as oil, as will beelaborated hereinbelow. Specifically, the ethyl or other esters of themicrobial oil can be enriched in the desired PUFA and separated bymethods commonly used in the art.

The term “eicosapentaenoic acid concentrate” or “EPA concentrate” is anoil concentrate and refers to an omega-3 oil comprising at least 70 wt %of EPA, measured as a wt % of oil, and substantially free of DHA. TheEPA concentrate is obtained from a microbial oil comprising 30 to 70 wt% of EPA, measured as a wt % of total fatty acids, and substantiallyfree of DHA, wherein said microbial oil is obtained from anoil-containing microbe that accumulates in excess of 25% of its dry cellweight as oil. The at least 70 wt % of EPA will be in the form of freefatty acids, triglycerides (e.g., TAGs), esters, and combinationsthereof. The esters are most preferably in the form of ethyl esters.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and are so called because at cellular pH, thelipids bear no charged groups. Generally, they are completely non-polarwith no affinity for water. Neutral lipids generally refer to mono-,di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol (MAG), diacylglycerol (DAG) or triacylglycerol (TAG),respectively, or collectively, acylglycerols. A hydrolysis reaction mustoccur to release FFAs from acylglycerols.

The term “triacylglycerols” is synonymous with the term“triacylglycerides” and refers to neutral lipids composed of three fattyacyl residues esterified to a glycerol molecule. TAGs can contain longchain PUFAs and saturated fatty acids, as well as shorter chainsaturated and unsaturated fatty acids. In living organisms, TAGs are theprimary storage units for fatty acids since the glycerol backbone helpsto stabilize PUFA molecules for storage or during transport. Incontrast, free fatty acids are rapidly oxidized.

The term “total fatty acids” (TFAs) herein refer to the sum of allcellular fatty acids that can be derivatized to fatty acid methyl esters(FAMEs) by the base transesterification method (as known in the art) ina given sample, which may be the biomass or oil, for example. Thus,total fatty acids include fatty acids from neutral lipid fractions(including DAGs, MAGs and TAGs) and from polar lipid fractions(including the phosphatidylcholine and the phosphatidylethanolaminefractions) but not FFAs.

The term “total lipid content” of cells is a measure of TFAs as apercent of the dry cell weight (DCW), although total lipid content canbe approximated as a measure of FAMEs as a percent of the DCW (FAMEs %DCW). Thus, total lipid content (TFAs % DCW) is equivalent to, e.g.,milligrams of total fatty acids per 100 milligrams of DCW.

The concentration of a fatty acid in the total lipid is expressed hereinas a weight percent of TFAs (% TFAs), e.g., milligrams of the givenfatty acid per 100 milligrams of TFAs. Unless otherwise specificallystated in the disclosure herein, reference to the percent of a givenfatty acid with respect to total lipids in microbial cells and inmicrobial oil is equivalent to concentration of the fatty acid as % TFAs(e.g., % EPA of total lipids is equivalent to EPA TFAs).

The concentration of a fatty acid ester (and/or fatty acid and/ortriglyceride, respectively) in an oil concentrate is expressed as aweight percent of oil [“% oil”], e.g. milligrams of the given fatty acidester (and/or fatty acid and/or triglyceride, respectively) per 100milligrams of oil concentrate. This unit of measurement is used todescribe the concentration of e.g., EPA in an EPA concentrate.

In some cases, it is useful to express the content of a given fattyacid(s) in a cell as its weight percent of the dry cell weight (% DCW).Thus, for example, eicosapentaenoic acid % DCW would be determinedaccording to the following formula: (eicosapentaenoic acid % TFAs)*(TFAs% DCW)]/100. The content of a given fatty acid(s) in a cell as itsweight percent of the dry cell weight (% DCW) can be approximated,however, as: (eicosapentaenoic acid % TFAs)*(FAMEs % DCW)]/100.

The terms “lipid profile” and “lipid composition” are interchangeableand refer to the amount of individual fatty acids contained in aparticular lipid fraction, such as in the total lipid or the oil,wherein the amount is expressed as a weight percent of TFAs. The sum ofthe individual fatty acids present in the mixture should be 100.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain lengths, from about C₁₂ to C₂₂, although bothlonger and shorter chain-length acids are known. The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon [“C”] atoms in the particular fatty acid and Y is thenumber of double bonds. Additional details concerning thedifferentiation between “saturated fatty acids” versus “unsaturatedfatty acids”, “monounsaturated fatty acids” versus “polyunsaturatedfatty acids” (PUFAs), and “omega-6 fatty acids” (“ω-6” or “n-6”) versus“omega-3 fatty acids” (“ω-3” or “n-3”) are provided in U.S. Pat. No.7,238,482, which is hereby incorporated herein by reference.

Nomenclature used to describe PUFAs herein is given in Table 4. In thecolumn titled “Shorthand Notation”, the omega-reference system is usedto indicate the number of carbons, the number of double bonds and theposition of the double bond closest to the omega carbon, counting fromthe omega carbon, which is numbered 1 for this purpose. The remainder ofthe Table summarizes the common names of omega-3 and omega-6 fatty acidsand their precursors, the abbreviations that will be used throughout thespecification and the chemical name of each compound.

TABLE 4 Nomenclature of Polyunsaturated Fatty Acids and PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitic Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic — cis-9- 18:1octadecenoic Linoleic LA cis-9, 12- 18:2 omega-6 octadecadienoic Gamma-GLA cis-6, 9, 12- 18:3 omega-6 Linolenic octadecatrienoic EicosadienoicEDA cis-11,14- 20:2 omega-6 eicosadienoic Dihomo- DGLA cis-8, 11, 14-20:3 omega-6 Gamma- eicosatrienoic Linolenic Arachidonic ARA cis-5, 8,11, 14- 20:4 omega-6 eicosatetraenoic Alpha-Linolenic ALA cis-9, 12, 15-18:3 omega-3 octadecatrienoic Stearidonic STA cis-6, 9, 12, 15- 18:4omega-3 octadecatetraenoic Nonadeca- NDPA cis-5, 8, 11, 14, 17- 19:5omega-2 pentaenoic nonadecapentaenoic Eicosatrienoic ETrA cis-11, 14,17- 20:3 omega-3 eicosatrienoic Eicosatetraenoic ETA cis-8, 11, 14, 17-20:4 omega-3 eicosatetraenoic Eicosapentaenoic EPA cis-5, 8, 11, 14, 17-20:5 omega-3 eicosapentaenoic Heneicosa- HPA cis-6, 9, 12, 15, 18- 21:5omega-3 pentaenoic heneicosapentaenoic Docosatetraenoic DTA cis-7, 10,13, 16- 22:4 omega-3 docosatetraenoic Docosapentaenoic DPAn-6 cis-4, 7,10, 13, 16- 22:5 omega-6 docosapentaenoic Docosapentaenoic DPAn-3 cis-22:5 omega-3 7, 10, 13, 16, 19- docosapentaenoic Docosahexaenoic DHAcis-4, 7, 10, 13, 16, 22:6 omega-3 19-docosahexaenoic

The terms EPA, DHA, NDPA and HPA, respectively, as used in the presentdisclosure, will refer to the respective acid or derivatives of the acid(e.g., glycerides, esters, phospholipids, amides, lactones, salts or thelike), unless specifically mentioned otherwise. For example, “EPA-EE”will specifically refer to EPA ethyl ester.

NDPA and HPA are commonly found in fish oils. Concentrated EPA producedfrom fish oils will often contain these fatty acids as impurities in thefinal EPA composition (see, e.g., U.S. Pat. Appl. Pub. No. 2010-0278879and Intl. Appl. Pub. No. WO 2010/147994 A1).

The term “substantially free of DHA” means comprising no more than about0.05 weight percent of DHA. Thus, an EPA concentrate is substantiallyfree of DHA when the concentration of DHA (in the form of free fattyacids, triacylglycerols, esters, and combinations thereof) is no morethan about 0.05 wt % of DHA, measured as a wt % of the oil. Similarly, amicrobial oil is substantially free of DHA (in the form of free fattyacids, triacylglycerols, esters, and combinations thereof) when theconcentration of DHA is no more than about 0.05 wt % of DHA, measured asa wt % of TFAs.

The terms “substantially free of NDPA” and “substantially free of HPA”are comparable to the definition provided above for the term“substantially free of DHA”, although the fatty acid NDPA or HPA,respectively, is substituted for DHA.

The term “high-level PUFA production” refers to production of at leastabout 25% PUFAs in the total lipids of the microbial host, preferably atleast about 30% PUFAs in the total lipids, more preferably at leastabout 35% PUFAs in the total lipids, more preferably at least about 40%PUFAs in the total lipids, more preferably at least about 40-45% PUFAsin the total lipids, more preferably at least about 45-50% PUFAs in thetotal lipids, more preferably at least about 50-60% PUFAs, and mostpreferably at least about 60-70% PUFAs in the total lipids. Thestructural form of the PUFA is not limiting; thus, for example, thePUFAs may exist in the total lipids as FFAs or in esterified forms suchas acylglycerols, phospholipids, sulfolipids or glycolipids.

The term “oil-containing microbe” refers to a microorganism capable ofproducing a microbial oil. Thus, an oil-containing microbe may be yeast,algae, euglenoids, stramenopiles, fungi, or combinations thereof. Inpreferred embodiments, the oil-containing microbe is oleaginous.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of oil (Weete, In: Fungal Lipid Biochemistry,2^(nd) Ed., Plenum, 1980). Generally, the cellular oil of oleaginousmicroorganisms follows a sigmoid curve, wherein the concentration oflipid increases until it reaches a maximum at the late logarithmic orearly stationary growth phase and then gradually decreases during thelate stationary and death phases (Yongmanitchai and Ward, Appl. Environ.Microbiol., 57:419-25 (1991)). It is not uncommon for oleaginousmicroorganisms to accumulate in excess of about 25% of their dry cellweight as oil.

Examples of oleaginous organisms include, but are not limited toorganisms from a genus selected from the group consisting ofMortierella, Thraustochytrium, Schizochytrium, Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.

The term “oleaginous yeast” refers to those oleaginous microorganismsclassified as yeasts that can make oil. Examples of oleaginous yeastinclude, but are by no means limited to, the following genera: Yarrowia,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces.

The term “pharmaceutical” as used herein means a compound or substancewhich, if sold in the United States, would be controlled by Section 503or 505 of the Federal Food, Drug and Cosmetic Act.

The term “substantially free of environmental pollutants” means the oilconcentrate or EPA concentrate, respectively, comprises either noenvironmental pollutants or at most only a trace of environmentalpollutants, wherein these include compounds such as polychlorinatedbiphenyls [“PCBs”] (CAS No. 1336-36-3), dioxins, brominated flameretardants and pesticides (e.g., toxaphenes anddichlorodiphenyltrichloroethane [“DDT”] and its metabolites).

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0) in oleaginous microorganisms, is described in detail inU.S. Pat. No. 7,238,482. Palmitate is the precursor of longer-chainsaturated and unsaturated fatty acid derivates, which are formed throughthe action of elongases and desaturases.

A wide spectrum of fatty acids (including saturated and unsaturatedfatty acids and short-chain and long-chain fatty acids) can beincorporated into TAGs, the primary storage unit for fatty acids. In theoil-containing microbes described herein, incorporation of long chainPUFAs into TAGs is most desirable, although the structural form of thePUFA is not limiting (thus, for example, EPA may exist in the totallipids as FFAs or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids). More specifically, in oneembodiment the oil-containing microbes will produce the at least onePUFA selected from the group consisting of LA, GLA, EDA, DGLA, ARA, DTA,DPAn-6, ALA, STA, ETrA, ETA, EPA, DPAn-3, DHA and mixtures thereof. Morepreferably, the at least one PUFA has at least a C₂₀ chain length, suchas PUFAs selected from the group consisting of EDA, DGLA, ARA, DTA,DPAn-6, ETrA, ETA, EPA, DPAn-3, DHA, and mixtures thereof.

In one embodiment, the at least one PUFA is selected from the groupconsisting of ARA, EPA, DPAn-6, DPAn-3, DHA and mixtures thereof. Inanother preferred embodiment, the at least one PUFA is selected from thegroup consisting of EPA and DHA.

Most PUFAs are incorporated into TAGs as neutral lipids and are storedin lipid bodies. However, it is important to note that a measurement ofthe total PUFAs within an oleaginous organism should minimally includethose PUFAs that are located in the phosphatidylcholine,phosphatidylethanolamine and TAG fractions.

The present invention relates to a method comprising:

-   -   (a) pelletizing a microbial biomass having a moisture level and        comprising oil-containing microbes;    -   (b) extracting the pelletized microbial biomass of step (a) to        produce an extracted oil; and,    -   (c) distilling the extracted oil of step (b) at least once under        short path distillation conditions, wherein said distillation        produces a distillate fraction and a lipid-containing fraction.        In another embodiment, the method set forth above may further        comprise the following steps:    -   (d) transesterifying the lipid-containing fraction of step (c);        and,    -   (e) enriching the transesterified lipid-containing fraction of        step (d) to obtain an oil concentrate.

Although the present invention is drawn to a process to obtain alipid-containing fraction or oil concentrate from pelletized microbialbiomass, the related processes that may be useful to obtain theoil-containing microbes themselves are also set forth in the schematicdiagram of FIG. 5. Each of the aspects of FIG. 5 will be discussed infurther detail below, with bold text herein referring to specificportions of FIG. 5.

Oil-containing microbes produce microbial biomass as the microbes growand multiply, typically via microbial fermentation. The microbialbiomass may be from any microorganism, whether naturally occurring orrecombinant (“genetically engineered”), capable of producing a microbialoil. Thus, for example, oil-containing microbes may be selected from thegroup consisting of yeast, algae, euglenoids, stramenopiles, fungi, andmixtures thereof. Preferably, the microorganism will be capable of highlevel PUFA production within the microbial oil.

As an example, commercial sources of ARA oil are typically produced frommicroorganisms in the genera Mortierella (filamentous fungus),Entomophthora, Pythium and Porphyridium (red alga). Most notably, MartekBiosciences Corporation (Columbia, Md.) produces an ARA-containingfungal oil (ARASCO®; U.S. Pat. No. 5,658,767) which is substantiallyfree of EPA and which is derived from either Mortierella alpina orPythium insidiuosum.

Similarly, EPA can be produced microbially via numerous differentprocesses based on the natural abilities of the specific microbialorganism utilized [e.g., heterotrophic diatoms Cyclotella sp. andNitzschia sp. (U.S. Pat. No. 5,244,921); Pseudomonas, Alteromonas orShewanella species (U.S. Pat. No. 5,246,841); filamentous fungi of thegenus Pythium (U.S. Pat. No. 5,246,842); Mortierella elongata, M.exigua, or M. hygrophila (U.S. Pat. No. 5,401,646); andeustigmatophycean alga of the genus Nannochloropsis (Krienitz, L. and M.Wirth, Limnologica, 36:204-210 (2006))].

DHA can also be produced using processes based on the natural abilitiesof native microbes. See, e.g., processes developed for Schizochytriumspecies (U.S. Pat. No. 5,340,742; U.S. Pat. No. 6,582,941); Ulkenia(U.S. Pat. No. 6,509,178); Pseudomonas sp. YS-180 (U.S. Pat. No.6,207,441); Thraustochytrium genus strain LFF1 (U.S. 2004/0161831 A1);Crypthecodinium cohnii (U.S. Pat. Appl. Pub. No. 2004/0072330 A1; deSwaaf, M. E. et al., Biotechnol. Bioeng., 81(6):666-672 (2003) and Appl.Microbiol. Biotechnol., 61(1):40-43 (2003)); Emiliania sp. (JapanesePatent Publication (Kokai) No. 5-308978 (1993)); and Japonochytrium sp.(ATCC #28207; Japanese Patent Publication (Kokai) No. 199588/1989)].Additionally, the following microorganisms are known to have the abilityto produce DHA: Vibrio marinus (a bacterium isolated from the deep sea;ATCC #15381); the micro-algae Cyclotella cryptica and Isochrysisgalbana; and, flagellate fungi such as Thraustochytrium aureum (ATCC#34304; Kendrick, Lipids, 27:15 (1992)) and the Thraustochytrium sp.designated as ATCC #28211, ATCC #20890 and ATCC #20891. Currently, thereare at least three different fermentation processes for commercialproduction of DHA: fermentation of C. cohnii for production of DHASCO™(Martek Biosciences Corporation, Columbia, Md.); fermentation ofSchizochytrium sp. for production of an oil formerly known as DHAGold(Martek Biosciences Corporation); and fermentation of Ulkenia sp. forproduction of DHActive™ (Nutrinova, Frankfurt, Germany).

Microbial production of PUFAs in microbial oils using recombinant meansis expected to have several advantages over production from naturalmicrobial sources. For example, recombinant microbes having preferredcharacteristics for oil production can be used, since the naturallyoccurring microbial fatty acid profile of the host can be altered by theintroduction of new biosynthetic pathways in the host and/or by thesuppression of undesired pathways, thereby resulting in increased levelsof production of desired PUFAs (or conjugated forms thereof) anddecreased production of undesired PUFAs. Secondly, recombinant microbescan provide PUFAs in particular forms which may have specific uses.Additionally, microbial oil production can be manipulated by controllingculture conditions, notably by providing particular substrate sourcesfor microbially expressed enzymes, or by addition of compounds/geneticengineering to suppress undesired biochemical pathways. Thus, forexample, it is possible to modify the ratio of omega-3 to omega-6 fattyacids so produced, or engineer production of a specific PUFA (e.g., EPA)without significant accumulation of other PUFA downstream or upstreamproducts.

Thus, for example, a microbe lacking the natural ability to make EPA canbe engineered to express a PUFA biosynthetic pathway by introduction ofappropriate PUFA biosynthetic pathway genes, such as specificcombinations of delta-4 desaturases, delta-5 desaturases, delta-6desaturases, delta-12 desaturases, delta-15 desaturases, delta-17desaturases, delta-9 desaturases, delta-8 desaturases, delta-9elongases, C_(14/16) elongases, C_(16/18) elongases, C_(18/20) elongasesand C_(20/22) elongases, although it is to be recognized that thespecific enzymes (and genes encoding those enzymes) introduced are by nomeans limiting to the invention herein.

As an example, several yeast organisms have been recombinantlyengineered to produce at least one PUFA. See for example, work inSaccharomyces cerevisiae (Dyer, J. M. et al., Appl. Eniv. Microbiol.,59:224-230 (2002); Domergue, F. et al., Eur. J. Biochem., 269:4105-4113(2002); U.S. Pat. No. 6,136,574; U.S. Pat. Appl. Pub. No.2006-0051847-A1) and the oleaginous yeast, Yarrowia lipolytica (U.S.Pat. No. 7,238,482; U.S. Pat. No. 7,465,564; U.S. Pat. No. 7,588,931;U.S. Pat. No. 7,932,077; U.S. Pat. No. 7,550,286; U.S. Pat. Appl. Pub.No. 2009-0093543-A1; U.S. Pat. Appl. Pub. No. 2010-0317072-A1).

In some embodiments, advantages are perceived if the microbial hostcells are oleaginous. Oleaginous yeast are naturally capable of oilsynthesis and accumulation, wherein the total oil content can comprisegreater than about 25% of the cellular dry weight, more preferablygreater than about 30% of the cellular dry weight, and most preferablygreater than about 40% of the cellular dry weight. In alternateembodiments, a non-oleaginous yeast can be genetically modified tobecome oleaginous such that it can produce more than 25% oil of thecellular dry weight, e.g., yeast such as Saccharomyces cerevisiae (IntlAppl. Pub. No. WO 2006/102342).

Genera typically identified as oleaginous yeast include, but are notlimited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oil-synthesizing yeasts include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis, and Yarrowia lipolytica (formerly classified asCandida lipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.,82(1):43-49 (2002)).

In some embodiments, it may be desirable for the oleaginous yeast to becapable of “high-level PUFA production”, wherein the organism canproduce at least about 5-10% of the desired PUFA (i.e., LA, ALA, EDA,GLA, STA, ETrA, DGLA, ETA, ARA, DPA n-6, EPA, DPA n-3 and/or DHA) in thetotal lipids. More preferably, the oleaginous yeast will produce atleast about 10-70% of the desired PUFA(s) in the total lipids. Althoughthe structural form of the PUFA is not limiting, preferably, TAGscomprise the PUFA(s).

Thus, the PUFA biosynthetic pathway genes and gene products describedherein may be produced in wildtype microbial host cells or heterologousmicrobial host cells, particularly in the cells of oleaginous yeasts(e.g., Yarrowia lipolytica). Expression in recombinant microbial hostsmay be useful for the production of various PUFA pathway intermediates,or for the modulation of PUFA pathways already existing in the host forthe synthesis of new products heretofore not possible using the host.

Although numerous oleaginous yeast could be engineered for production ofpreferred omega-3/omega-6 PUFAs based on the cited teachings providedabove, representative PUFA-producing strains of the oleaginous yeastYarrowia lipolytica are described in Table 5. These strains possessvarious combinations of the following PUFA biosynthetic pathway genes:delta-4 desaturases, delta-5 desaturases, delta-6 desaturases, delta-12desaturases, delta-15 desaturases, delta-17 desaturases, delta-9desaturases, delta-8 desaturases, delta-9 elongases, C_(14/16)elongases, C_(16/18) elongases, C_(18/20) elongases and C_(20/22)elongases, although it is to be recognized that the specific enzymes(and genes encoding those enzymes) introduced and the specific PUFAsproduced are by no means limiting to the invention herein.

TABLE 5 Lipid Profiles of Representative Yarrowia lipolytica StrainsEngineered to Produce Omega-3/Omega-6 PUFAs ATCC Fatty Acid Content (AsA Percent [%] of Total Fatty Acids) TFAs Deposit 18:3 20:2 DPA % StrainReference No. 16:0 16:1 18:0 18:1 18:2 (ALA) GLA (EDA) DGLA ARA ETA EPAn-3 DHA DCW Wild- U.S. #76982 14 11 3.5 34.8 31 0  0 — — — — — — — —type Pat. No. pDM- 7,465,564 — 11.9  8.6 1.5 24.4 17.8 0 25.9 — — — — —— — — W208 pDMW- — 16.2  1.5 0.1 17.8 22.2 0 34 — — — — — — — — 208-D62M4 U.S. — 15  4 2  5 27 0 35 —  8  0  0  0 — — — Pat. No. 7,932,077Y2034 U.S. — 13.1  8.1 1.7  7.4 14.8 0 25.2 —  8.3 11.2 — — — — — Pat.No. Y2047 7,588,931 PTA- 15.9  6.6 0.7  8.9 16.6 0 29.7 —  0 10.9 — — —— — 7186 Y2214 —  7.9 15.3 0 13.7 37.5 0  0 —  7.9 14 — — — — — EU U.S.— 19 10.3 2.3 15.8 12 0 18.7 —  5.7  0.2 3 10.3 — — 36 Y2072 Pat. No. — 7.6  4.1 2.2 16.8 13.9 0 27.8 —  3.7  1.7 2.2 15 — — — Y2102 7,932,077—  9  3 3.5  5.6 18.6 0 29.6 —  3.8  2.8 2.3 18.4 — — — Y2088 — 17  4.53  2.5 10 0 20 —  3  2.8 1.7 20 — — — Y2089 —  7.9  3.4 2.5  9.9 14.3 037.5 —  2.5  1.8 1.6 17.6 — — — Y2095 — 13  0 2.6  5.1 16 0 29.1 —  3.1 1.9 2.7 19.3 — — — Y2090 —  6  1 6.1  7.7 12.6 0 26.4 —  6.7  2.4 3.626.6 — — 22.9 Y2096 PTA-  8.1  1 6.3  8.5 11.5 0 25 —  5.8  2.1 2.5 28.1— — 20.8 7184 Y2201 PTA- 11 16.1 0.7 18.4 27 0 —  3.3  3.3  1 3.8  9 — —— 7185 Y3000 U.S. PTA-  5.9  1.2 5.5  7.7 11.7 0 30.1    2.6  1.2 1.2 4.7 18.3 5.6 — Pat. No. 7187 7,550,286 Y4001 U.S. —  4.3  4.4 3.9 35.923 0 — 23.8 0  0 0 — — — — Y4036 Pat. —  7.7  3.6 1.1 14.2 32.6 0 — 15.618.2  0 0 — — — — Y4070 application —  8  5.3 3.5 14.6 42.1 0 —  6.7 2.4 11.9 — — — — — Y4086 Pub. —  3.3  2.2 4.6 26.3 27.9 6.9 —  7.6  1 0 2  9.8 — — 28.6 Y4128 No. PTA-  6.6  4 2  8.8 19 2.1 —  4.1  3.2  05.7 42.1 — — 18.3 2009- 8614 Y4158 0093543- —  3.2  1.2 2.7 14.5 30.45.3 —  6.2  3.1  0.3 3.4 20.5 — — 27.3 Y4184 A1 —  3.1  1.5 1.8  8.731.5 4.9 —  5.6  2.9  0.6 2.4 28.9 — — 23.9 Y4217 —  3.9  3.4 1.2  6.219 2.7 —  2.5  1.2  0.2 2.8 48.3 — — 20.6 Y4259 —  4.4  1.4 1.5  3.919.7 2.1 —  3.5  1.9  0.6 1.8 46.1 — — 23.7 Y4305 —  2.8  0.7 1.3  4.917.6 2.3 —  3.4  2  0.6 1.7 53.2 — — 27.5 Y4127 Int'l. PTA-  4.1  2.32.9 15.4 30.7 8.8 —  4.5  3.0  3.0 2.8 18.1 — — — App. 8802 Y4184 Pub. — 2.2  1.1 2.6 11.6 29.8 6.6 —  6.4  2.0  0.4 1.9 28.5 — — 24.8 Y8404 No.WO —  2.8  0.8 1.8  5.1 20.4 2.1  2.9  2.5  0.6 2.4 51.1 — — 27.3 Y84062008/ PTA-  2.6  0.5 2.9  5.7 20.3 2.8  2.8  2.1  0.5 2.1 51.2 — — 30.7073367 10025 Y8412 PTA-  2.5  0.4 2.6  4.3 19.0 2.4  2.2  2.0  0.5 1.955.8 — — 27.0 10026 Y8647 —  1.3  0.2 2.1  4.7 20.3 1.7  3.3  3.6  0.73.0 53.6 — — 37.6 Y8649 —  2.4  0.3 2.9  3.7 18.8 2.2  2.1  2.4  0.6 2.155.8 — — 27.9 Y8650 U.S. Pat. —  2.2  0.3 2.9  3.8 18.8 2.4  2.1  2.4 0.6 2.1 56.1 — — 28.2 Y9028 application —  1.3  0.2 2.1  4.4 19.8 1.7 3.2  2.5  0.8 1.9 54.5 — — 39.6 Y9031 Pub. —  1.3  0.3 1.8  4.7 20.11.7  3.2  3.2  0.9 2.6 52.3 — — 38.6 Y9477 No. 2010- —  2.6  0.5 3.4 4.8 10.0 0.5  2.5  3.7  1.0 2.1 61.4 — — 32.6 Y9497 0317072- —  2.4 0.5 3.2  4.6 11.3 0.8  3.1  3.6  0.9 2.3 58.7 — — 33.7 Y9502 A1 —  2.5 0.5 2.9  5.0 12.7 0.9  3.5  3.3  0.8 2.4 57.0 — — 37.1 Y9508 —  2.3 0.5 2.7  4.4 13.1 0.9  2.9  3.3  0.9 2.3 58.7 — — 34.9 Y8143 —  4.2 1.5 1.4  3.6 18.1 2.6  1.7  1.6  0.6 1.6 50.3 — — 22.3 Y8145 —  4.3 1.7 1.4  4.8 18.6 2.8  2.2  1.5  0.6 1.5 48.5 — — 23.1 Y8259 PTA-  3.5 1.3 1.3  4.8 16.9 2.3  1.9  1.7  0.6 1.6 53.9 — — 20.5 10027   Y8367 — 3.7  1.2 1.1  3.4 14.2 1.1  1.5  1.7  0.8 1.0 58.3 — — 18.4 Y8370 — 3.4  1.1 1.4  4.0 15.7 1.9  1.7  1.9  0.6 1.5 56.4 — — 23.3 Y8670 — 1.9  0.4 3.4  4.3 17.0 1.5  2.2  1.7  0.6 1.1 60.9 — — 27.3 Y8672 — 2.3  0.4 2.0  4.0 16.1 1.4  1.8  1.6  0.7 1.1 61.8 — — 26.5

One of skill in the art will appreciate that the methodology of thepresent invention is not limited to the Yarrowia lipolytica strainsdescribed above, nor to the species (i.e., Yarrowia lipolytica) or genus(i.e., Yarrowia) in which the invention has been demonstrated, as themeans to introduce a PUFA biosynthetic pathway into an oleaginous yeastare well known. Instead, any oleaginous yeast or any other suitablemicrobe capable of producing microbial oils (preferably comprisingPUFAs, e.g., LA, GLA, EDA, DGLA, ARA, DTA, DPAn-6, ALA, STA, ETrA, ETA,EPA, DPAn-3, DHA) will be equally suitable for use in the presentmethodologies, as demonstrated in Example 26 (although some processoptimization may be required for each new microbe handled, based ondifferences in, e.g., the cell wall composition of each oil-containingmicrobe).

A microbial species producing a lipid, preferably comprising a PUFA(s),may be cultured and grown in a fermentation medium under conditionswhereby the lipid is produced by the microorganism. Typically, themicroorganism is fed with a carbon and nitrogen source, along with anumber of additional chemicals or substances that allow growth of themicroorganism and/or production of the microbial oil (preferablycomprising PUFAs). The fermentation conditions will depend on themicroorganism used, as described in the above citations, and may beoptimized for a high content of the PUFA(s) in the resulting biomass.

In general, media conditions may be optimized by modifying the type andamount of carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the amount of different mineral ions, theoxygen level, growth temperature, pH, length of the biomass productionphase, length of the oil accumulation phase and the time and method ofcell harvest. For example, Yarrowia lipolytica are generally grown in acomplex media such as yeast extract-peptone-dextrose broth (YPD) or adefined minimal media (e.g., Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.) that lacks a component necessary for growth and therebyforces selection of the desired recombinant expression cassettes thatenable PUFA production).

When the desired amount of microbial oil, preferably comprising PUFAs,has been produced by the microorganism, the fermentation medium may bemechanically processed to obtain untreated microbial biomass comprisingthe microbial oil. For example, the fermentation medium may be filteredor otherwise treated to remove at least part of the aqueous component(e.g., by drying). As will be appreciated by those in the art, theuntreated microbial biomass typically includes water. Preferably, aportion of the water is removed from the untreated microbial biomassafter microbial fermentation to provide a microbial biomass with amoisture level of less than 10 weight percent, more preferably amoisture level of less than 5 weight percent, and most preferably amoisture level of 3 weight percent or less. The microbial biomassmoisture level can be controlled in drying. Preferably the microbialbiomass has a moisture level in the range of about 1 to 10 weightpercent.

Optionally, the fermentation medium and/or the microbial biomass may bepasteurized or treated via other means to reduce the activity ofendogenous microbial enzymes that can harm the microbial oil and/or PUFAproducts.

Thus, the microbial biomass may be in the form of whole cells, wholecell lysates, homogenized cells, partially hydrolyzed cellular material,and/or disrupted cells (i.e., disrupted microbial biomass).

The microbial biomass may be mechanically processed to disrupting thebiomass, for example via cellular lysing or via physical means such asbead beaters, screw extrusion, etc. to provide greater accessibility tothe cell contents.

The disrupted microbial biomass will have a disruption efficiency of atleast 50% of the oil-containing microbes. More preferably, thedisruption efficiency is at least 75%, more preferably at least 80% andmost preferably 85-90% or more, of the oil-containing microbes. Althoughpreferred ranges are described above, useful examples of disruptionefficiencies include any integer percentage from 50% to 100%, such as51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% disruption efficiency.

The disruption efficiency refers to the percent of cells walls that havebeen fractured or ruptured during processing, as determinedqualitatively by optical visualization or as determined quantitativelyaccording to the following formula: % disruption efficiency=(% freeoil*100) divided by (% total oil), wherein % free oil and % total oilare measured for the solid pellet.

A solid pellet that has been not subjected to a process of disruption(e.g., mechanical crushing using e.g., screw extrusion, an expeller,pistons, bead beaters, mortar and pestle, Hammer-milling, air-jetmilling, etc.) will typically have a low disruption efficiency sincefatty acids within DAGs, MAGs and TAGs, phosphatidylcholine andphosphatidylethanolamine fractions and free fatty acids, etc. aregenerally not extractable from the microbial biomass until a process ofdisruption has broken both cell walls and internal membranes of variousorganelles, including membranes surrounding lipid bodies. Variousprocesses of disruption will result in various disruption efficiencies,based on the particular shear, compression, static and dynamic forcesinherently produced in the process.

Increased disruption efficiency of the microbial biomass typically leadsto increased extraction yields (e.g., as measured by the weight percentof crude extracted oil), likely since more of the microbial oil issusceptible to the presence of the extraction solvent(s) with disruptionof cell walls and membranes.

Although a variety of equipment may be utilized to produce the disruptedmicrobial biomass, preferably the disrupting is performed in a twinscrew extruder. More specifically, the twin screw extruder preferablycomprises: (i) a total specific energy input (SEI) in the extruder ofabout 0.04 to 0.4 KW/(kg/hr), more preferably 0.05 to 0.2 KW/(kg/hr) andmost preferably about 0.07 to 0.15 KW/(kg/hr); (ii) a compaction zoneusing bushing elements with progressively shorter pitch length; and,(iii) a compression zone using flow restriction. Most of the mechanicalenergy required for cell disruption is imparted in the compression zone,which is created using flow restriction in the form of e.g., reversescrew elements, restriction/blister ring elements or kneading elements.The compaction zone is prior to the compression zone within theextruder. A first zone of the extruder may be present to feed andtransport the biomass into the compaction zone.

The process of pelletization generally involves the following steps: (1)mixing the microbial biomass and at least one grinding agent capable ofabsorbing oil to provide a disrupted biomass mix comprising disruptedmicrobial biomass; (2) blending the disrupted biomass mix with at leastone binding agent to provide a fixable mix capable of forming a solidpellet; and, (3) forming said fixable mix into solid pellets to providea pelletized microbial biomass.

First, the microbial biomass, having a moisture level and comprisingoil-containing microbes, is mixed with at least one grinding agentcapable of absorbing oil, to provide a disrupted biomass mix.

The grinding agent, capable of absorbing oil, may be a particle having aMoh hardness of 2.0 to 6.0, and preferably 2.0 to about 5.0; and morepreferably about 2.0 to 4.0; and an oil absorption coefficient of 0.8 orhigher, preferably 1.0 or higher, and more preferably 1.3 or higher, asdetermined according to the American Society for Testing And Materials(ASTM) Method D1483-60. Preferred grinding agents have a median particlediameter of about 2 to 20 microns, and preferably about 7 to 10 microns;and a specific surface area of at least 1 m²/g and preferably 2 to 100m²/g as determined with the BET method (Brunauer, S. et al., J. Am.Chem. Soc., 60:309 (1938)).

Preferred grinding agents are selected from the group consisting ofsilica and silicate. As used herein, the term “silica” refers to a solidchemical substance consisting mostly (at least 90% and preferably atleast 95% by weight) of silicon and oxygen atoms in a ratio of about twooxygen atoms to one silicon atom, thus having the empirical formula ofSiO₂. Silicas include, for example, precipitated silicas, fumed silicas,amorphous silicas, diatomaceous silicas, also known as diatomaceousearths (D-earth) as well as silanized forms of these silicas. The term“silicate” refers to a solid chemical substance consisting mostly (atleast 90% and preferably at least 95% by weight) of atoms of silicon,oxygen and at least one metal ion. The metal ion may be, for instance,lithium, sodium, potassium, magnesium, calcium, aluminum, or a mixturethereof. Aluminum silicates in the form of zeolites, natural andsynthetic, may be used. Other silicates that may be useful are calciumsilicates, magnesium silicates, sodium silicates, and potassiumsilicates.

A preferred grinding agent is diatomaceous earth (D-earth) having aspecific surface area of about 10-20 m²/g and an oil absorptioncoefficient of 1.3 or higher. A commercial source of a suitable grindingagent capable of absorbing oil is Celite 209 D-earth available fromCelite Corporation, Lompoc, Calif.

Other grinding agents may be poly(meth)acrylic acids, and ionomersderived from partial or full neutralization of poly(meth)acrylic acidswith sodium or potassium bases. Herein the term (meth)acrylate means thecompound may be either an acrylate, a methacrylate, or a mixture of thetwo.

The at least one grinding agent is present at about 1 to 20 weightpercent, and more preferably 1 to 15 weight percent, and most preferablyabout 2 to 12 weight percent, based on the summation of components (a)microbial biomass, (b) grinding agent and (c) binding agent in the solidpellet.

Mixing a microbial biomass and a grinding agent capable of absorbing oilto provide a disrupted biomass mix [step (1)] can be performed by anymethod known in the art to apply energy to a mixing media. Preferablythe mixing provides a disrupted biomass mix having a temperature of 90°C. or less, and more preferably 70° C. or less.

For example, the microbial biomass and grinding agent may be fed into amixer, such as a single screw extruder or twin screw extruder, agitator,single screw or twin screw kneader, or Banbury mixer, and the additionstep may be addition of all ingredients at once or gradual addition inbatches.

Preferably the mixing is performed in a twin screw extruder, asdescribed above, having a SEI of about 0.04 to 0.4 KW/(kg/hr), acompaction zone using bushing elements with progressively shorter pitchlength, and a compression zone using flow restriction. Under theseconditions, the initial microbial biomass may be whole dried cells andthe process of cell disruption, resulting in a disrupted microbialbiomass having a disruption efficiency of at least 50% of theoil-containing microbes, may occur at the beginning or during the mixingstep, that is, cell disruption and step (1) may be combined andsimultaneous to produce a disrupted biomass mix. The presence of thegrinding agent enhances cell disruption; however, most cell disruptionoccurs as a result of the twin screw extruder itself.

Thus, for clarity, cell disruption of the microbial biomass can beperformed in the absence of grinding agent, for instance in a twin screwextruder having a compression zone as disclosed above and then mixing ofgrinding agent and disrupted microbial biomass can be performed in thetwin screw extruder or a variety of other mixers to provide thedisrupted biomass mix. Or, cell disruption of the microbial biomass canbe performed in the presence of grinding agent, for instance in a twinscrew extruder having a compression zone. In either case, however, celldisruption (i.e., disruption efficiency) should be maximized if onedesires to maximize the yield of extracted oil from the oil-containingmicrobes in subsequent process steps.

At least one binding agent is then blended with the disrupted biomassmix to provide a fixable mix capable of forming a solid pellet (i.e.,the pelletized microbial biomass).

Binding agents useful in the invention include hydrophilic organicmaterials and hydrophilic inorganic materials that are water soluble orwater dispersible. Preferred water soluble binding agents havesolubility in water of at least 1 weight percent, preferably at least 2weight percent and more preferably at least 5 weight percent, at 23° C.

The binding agent preferably has solubility in supercritical fluidcarbon dioxide at 500 bar and 40° C. of less than 1×10⁻³ mol fraction;and preferably less than 1×10⁻⁴, more preferably less than 1×10⁻⁵, andmost preferably less than 1×10⁻⁶ mol fraction. The solubility may bedetermined according to the methods disclosed in “Solubility inSupercritical Carbon Dioxide”, Ram Gupta and Jae-Jin Shim, Eds., CRC(2007).

The binding agent acts to retain the integrity and size of pelletsformed from the pelletization process and furthermore acts to reducefines in further processing and transport of the pellets.

Suitable organic binding agents include: alkali metal carboxymethylcellulose with degrees of substitution of 0.5 to 1; polyethylene glycoland/or alkyl polyethoxylate, preferably with an average molecular weightbelow 1,000; phosphated starches; cellulose and starch ethers, such ascarboxymethyl starch, methyl cellulose, hydroxyethyl cellulose,hydroxypropyl cellulose and corresponding cellulose mixed ethers;proteins including gelatin and casein; polysaccharides includingtragacanth, sodium and potassium alginate, guam Arabic, tapioca, partlyhydrolyzed starch including maltodextrose and dextrin, and solublestarch; sugars including sucrose, invert sugar, glucose syrup andmolasses; synthetic water-soluble polymers includingpoly(meth)acrylates, copolymers of acrylic acid with maleic acid orcompounds containing vinyl groups, polyvinyl alcohol, partiallyhydrolyzed polyvinyl acetate and polyvinyl pyrrolidone. If the compoundsmentioned above are those containing free carboxyl groups, they arenormally present in the form of their alkali metal salts, moreparticularly their sodium salts.

Phosphated starch is understood to be a starch derivative in whichhydroxyl groups of the starch anhydroglucose units are replaced by thegroup —O—P(O)(OH)₂ or water-soluble salts thereof, more particularlyalkali metal salts, such as sodium and/or potassium salts. The averagedegree of phosphation of the starch is understood to be the number ofesterified oxygen atoms bearing a phosphate group per saccharide monomerof the starch averaged over all the saccharide units. The average degreeof phosphation of preferred phosphate starches is in the range from 1.5to 2.5.

Partly hydrolyzed starches in the context of the present invention areunderstood to be oligomers or polymers of carbohydrates which may beobtained by partial hydrolysis of starch using conventional, for exampleacid- or enzyme-catalyzed processes. The partly hydrolyzed starches arepreferably hydrolysis products with average molecular weights of 440 to500,000. Polysaccharides with a dextrose equivalent (DE) of 0.5 to 40and, more particularly, 2 to 30 are preferred, DE being a standardmeasure of the reducing effect of a polysaccharide by comparison withdextrose (which has a DE of 100, i.e., DE 100). Both maltodextrins (DE3-20) and dry glucose syrups (DE 20-37) and also so-called yellowdextrins and white dextrins with relatively high average molecularweights of about 2,000 to 30,000 may be used after phosphation.

A preferred class of binding agent is water and carbohydrates selectedfrom the group consisting of sucrose, lactose, fructose, glucose, andsoluble starch. Preferred binding agents have a melting point of atleast 50° C., preferably at least 80° C., and more preferably at least100° C.

Suitable inorganic binding agents include sodium silicate, bentonite,and magnesium oxide.

Preferred binding agents are materials that are considered “food grade”or “generally recognized as safe” (GRAS).

The binding agent is present at about 0.5 to 10 weight percent,preferably 1 to 10 weight percent, and more preferably about 3 to 8weight percent, based on the summation of components (a) microbialbiomass, (b) grinding agent and (c) binding agent in the solid pellet.

As one of skill in the art will appreciate, fixable mix will havesignificantly higher moisture level than the moisture level of the finalsolid pellet, to permit ease of handling (e.g., extruding the fixablemix into a die). Thus, for example, a binding agent comprising asolution of sucrose and water can be added to the disrupted biomass mixin a manner that results in a fixable mix having within 0.5 to 20 weightpercent water. However, upon drying of the fixable mix to form a solidpellet, the final moisture level of the solid pellet is less than 5weight percent of water and the sucrose is less than 10 weight percent.

Blending the at least one binding agent with disrupted biomass mix toprovide a fixable mix [step (2)] can be performed by any method thatallows dissolution of the binding agent and blending with the disruptedbiomass mix to provide a fixable mix. The term “fixable mix” means thatthe mix is capable of forming a solid pellet upon removal of solvent,for instance water, in a drying step.

The binding agent can be blended by a variety of means. One methodincludes dissolution of the binding agent in a solvent to provide abinder solution, following by metering the binder solution, at acontrolled rate, into the disrupted biomass mix. A preferred solvent iswater, but other solvents, for instance ethanol, isopropanol, and such,may be used advantageously. Another method includes adding the bindingagent, as a solid or solution, to the biomass/grinding agent at thebeginning or during the mixing step, that is, step (1) and (2) arecombined and simultaneous. If the binding agent is added as a solid,preferably sufficient moisture is present in the disrupted biomass mixto dissolve the binding agent during the blending step. A preferredmethod of blending includes metering the binder solution, at acontrolled rate, into the disrupted biomass mix in an extruder,preferably after the compression zone, as disclosed above. The additionof a binder solution after the compression zone allows for rapid coolingof the disrupted biomass mix.

Forming solid pellets comprising pelletized microbial biomass from thefixable mix [step (3)] can be performed by a variety of means known inthe art. One method includes extruding the fixable mix into a die, forinstance a dome granulator, to form strands of uniform diameter that aredried on a vibrating or fluidized bed drier to break the strands toprovide pellets. The pelletized microbial biomass is suitable fordownstream oil extraction, transport, or other purposes.

The solid pellets disclosed herein desirably are non-tacky at roomtemperature. A large plurality of the solid pellets may be packedtogether for many days without degradation of the pellet structure, andwithout binding together. A large plurality of pellets desirably is afree-flowing pelletized composition. Preferably the pellets have anaverage diameter of about 0.5 to about 1.5 mm and an average length ofabout 2.0 to about 8.0 mm. Preferably, the solid pellets have a finalmoisture level of about 0.1% to 5.0%, with a range about 0.5% to 3.0%more preferred. Increased moisture levels in the final solid pellets maylead to difficulties during storage due to growth of e.g., molds.

The solid pellet therefore preferably comprises: (a) about 70 to about98.5 weight percent of disrupted biomass comprising oil-containingmicrobes; (b) about 1 to about 20 weight percent grinding agent capableof absorbing oil; and, (c) about 0.5 to 10 weight percent binding agent;based on the summation of (a), (b) and (c) in the solid pellet. Thesolid pellet may comprise 75 to 98 weight percent (a); 1 to 15 weightpercent (b) and 1 to 10 weight percent (c); and, preferably the pelletcomprises 80 to 95 weight percent (a); 2 to 12 weight percent (b) and 3to 8 weight percent (c).

The pelletization methodology set forth above has proven to beeffective, highly scale-able, robust and user-friendly, while allowingproduction at relatively high yields and at high throughput rates. Celldisruption using conventional techniques such as spray drying, use ofhigh shear mixers, etc. was found to be inadequate for e.g., yeast cellwalls comprising chitin. Incumbent wet media mill disruption processproduced fines and colloidal contamination which necessitated furtherseparation steps and resulted in significant oil loss. Additionally, wetmedia milling steps introduced a liquid carrier (e.g., isohexane orwater) which complicated downstream processing by requiring liquid-solidseparation step with oil losses. The pelletization process describedherein relies on the production of a disrupted biomass mix; however,advantageously, the disruption occurs without requiring a liquidcarrier. Furthermore, the presence of the grinding agent within thesolid pellets appears to facilitate high levels of oil extraction. And,since the pellets remain durable throughout the extraction process, thisaids operability and cycle time.

The pelletized microbial biomass is extracted with a solvent to providean extracted oil and an extracted pellet (i.e., the “residual biomass”or “residual pellet”).

Oil extraction can occur via treatment with various organic solvents(e.g., hexane, iso-hexane), enzymatic extraction, osmotic shock,ultrasonic extraction, supercritical fluid extraction (e.g., CO₂extraction), saponification and combinations of these methods.

In one embodiment, extraction is performed with an organic solvent toproduce an extracted oil and said extracted oil is degummed andoptionally bleached prior to said step (c) distilling the extracted oil.More specifically, the crude oil can be degummed by water or acidhydration of phospholipids and other polar and neutral lipid complexes,followed by separation of the precipitated gum from the oil.Alternatively, the phospholipids and other hydratable impurities can beremoved by contacting the oil with a polar solvent such as acetone orthrough enzymatic degumming. The degummed oil may be further bleachedusing bleaching clays, silica or carbons to remove color compounds andresidual metals etc.

In an alternate embodiment, extraction occurs using supercriticalconditions. Supercritical fluids (SCFs) exhibit properties intermediatebetween those of gases and liquids. A key feature of a SCF is that thefluid density can be varied continuously from liquid-like to gas-likedensities by varying either the temperature or pressure, or acombination thereof. Various density-dependent physical propertieslikewise exhibit similar continuous variation in this region. Some ofthese properties include, but are not limited to, solvent strength (asevidenced by the solubilities of various substances in the SCF media),polarity, viscosity, diffusivity, heat capacity, thermal conductivity,isothermal compressibility, expandability, contractibility, fluidity,and molecular packing. The density variation in a SCF also influencesthe chemical potential of solutes and hence, reaction rates andequilibrium constants. Thus, the solvent environment in a SCF media canbe optimized for a specific application by tuning the variousdensity-dependent fluid properties.

A fluid is in the SCF state when the system temperature and pressureexceed the corresponding critical point values defined by the criticaltemperature (T_(c)) and critical pressure (P_(c)). For pure substances,the T_(c) and P_(c) are the highest at which vapor and liquid phases cancoexist. Above the T_(c), a liquid does not form for a pure substance,regardless of the applied pressure. Similarly, the P_(c) and criticalmolar volume are defined at this T_(c) corresponding to the state atwhich the vapor and liquid phases merge. Although more complex formulticomponent mixtures, a mixture critical state is similarlyidentified as the condition at which the properties of coexisting vaporand liquid phases become indistinguishable. For a discussion ofsupercritical fluids, see Kirk-Othmer Encycl. of Chem. Technology,4^(th) ed., Vol. 23, pg. 452-477, John Wiley & Sons, NY (1997).

Any suitable SCF or liquid solvent may be used in the oil extractionstep, e.g., the contacting of the solid pellets with a solvent toseparate the oil from the microbial biomass, including, but not limitedto, CO₂, tetrafluoromethane, ethane, ethylene, propane, propylene,butane, isobutane, isobutene, pentane, hexane, cyclohexane, benzene,toluene, xylenes, and mixtures thereof, provided that it is inert to allreagents and products. Preferred solvents include CO₂ or a C₃-C₆ alkane.More preferred solvents are CO₂, pentane, butane, and propane. Mostpreferred solvents are supercritical fluid solvents comprising CO₂.

In a preferred embodiment, super-critical CO₂ extraction is performed,as disclosed in U.S. Pat. Pub. No. 2011-0263709-A1. By application ofthis particular methodology, the pelletized microbial biomass issubjected to supercritical oil extraction conditions. Phospholipids(PLs) remain within the residual biomass (i.e., the extracted residualpellet), while the resulting extract (i.e., an extract comprising alipid fraction substantially free of phospholipids) is fractionated atleast once to produce an extracted oil having a refined lipidcomposition that may comprise neutral lipids and/or free fatty acids(FFAs) while being substantially free of PLs. The refined lipidcomposition may be enriched in TAGs (preferably comprising PUFAs)relative to the oil composition of the pelletized microbial biomass thatwas not processed with the solvent. The refined lipid composition mayundergo further purification to produce a purified oil.

In this method, the supercritical fluids comprising CO₂ may furthercomprise at least one additional solvent (i.e., a cosolvent), forexample one or more of the solvents listed above, as long as thepresence or amount of the additional solvent is not deleterious to theprocess, for example does not solubilize the PLs contained in themicrobial biomass during the primary extraction step. However, a polarcosolvent such as ethanol, methanol, acetone, or the like may be addedto intentionally impart polarity to the solvent phase to enableextraction of the PLs from the microbial biomass during optionalsecondary oil extractions to isolate the PLs.

The solid pellets comprising oil-containing disrupted microbial biomassmay be contacted with liquid or supercritical CO₂ under suitableextraction conditions to provide an extracted oil and a residual biomassaccording to at least two methods. According to a first method of U.S.Pat. Appl. Pub. No. 2011-0263709-A1, contacting the pelletized microbialbiomass with CO₂ is performed multiple times under extraction conditionscorresponding to increasing solvent density, for example underincreasing pressure and/or decreasing temperature, to obtain extractscomprising a refined lipid composition wherein the lipid fractions aresubstantially free of PLs. The refined lipid composition of the extractsvaries in the distribution of FFAs, monoacylglycerols (MAGs),diacylglycerols (DAGs), and TAGs according to their relativesolubilities, which depend upon the solvent density corresponding to theselected extraction conditions of each of the multiple extractions.

Alternatively and according to the present methods, in a second methodof U.S. Pat. Appl. Pub. No. 2011-0263709-A1, the pelletized microbialbiomass is contacted with a solvent such as CO₂ under extractionconditions selected to provide an extract comprising a lipid fractionsubstantially free of PLs, which subsequently undergoes a series ofmultiple staged pressure letdown steps to provide refined lipidcompositions. Each of these staged pressure letdown steps is conductedin a separator vessel at pressure and temperature conditionscorresponding to decreasing solvent density to isolate a liquid-phaserefined lipid composition which can be separated from the extract phaseby, for example, simple decantation. The refined lipid compositionswhich are provided vary in the distribution of FFAs, MAGs, DAGs, andTAGs according to their relative solubilities, which depend upon thesolvent density corresponding to the selected conditions of the stagedseparator vessels.

The extracted oils having refined lipid compositions obtained using thesecond method described above may correspond to the extracts obtained inthe first method when extraction conditions are appropriately matched.It is thus believed possible to exemplify the refined lipid compositionsobtainable as described herein through performance of the first method.

According to the present methods, the solid pellets comprisingoil-containing disrupted microbial biomass may be contacted with asolvent such as liquid or SCF CO₂ at a temperature and pressure and fora contacting time sufficient to obtain an extract comprising a lipidfraction substantially free of PLs. The lipid fraction may compriseneutral lipids (e.g., comprising TAGs, DAGs, and MAGs) and FFAs. Thecontacting and fractionating temperatures may be chosen to provideliquid or SCF CO₂, to be within the thermal stability range of thePUFA(s), and to provide sufficient density of the CO₂ to solubilize theTAGs, DAGs, MAGs, and FFAs. Generally, the contacting and fractionatingtemperatures may be from about 20° C. to about 100° C., for example fromabout 35° C. to about 100° C.; the pressure may be from about 60 bar toabout 800 bar, for example from about 80 bar to about 600 bar. Asufficient contacting time, as well as appropriate CO₂ to microbialbiomass ratios, may be determined by generating extraction curves for aparticular sample of solid pellets. These extraction curves aredependent upon the extraction conditions of temperature, pressure, CO₂flow rate, and variables such as the extent of cell disruption and theform of the microbial biomass. In one embodiment of the present methods,the solvent comprises liquid or supercritical fluid CO₂ and the massratio of CO₂ to the microbial biomass is from about 20:1 to about 70:1,for example from about 20:1 to about 50:1.

The extract comprising a lipid fraction substantially free of PLs maythen be fractionated to obtain an extracted oil having a refined lipidcomposition comprising at least one PUFA, wherein the refined lipidcomposition is enriched in TAGs relative to the oil composition of thepelletized microbial biomass that is not processed with solvent. Therefined lipid composition may further comprise DAGs, MAGs, or acombination of these. The refined lipid composition may further compriseFFAs. Other refined lipid compositions which may be obtained separatelyor in combination in the fractionation step include a TAG enrichedproduct that is depleted in FFAs, a FFA enriched product that isdepleted in TAGs, a FFA enriched product that is enriched in MAGs and/orDAGs, a FFA enriched product that is depleted in MAGs and/or DAGs, a TAGenriched product that is enriched in MAGs and/or DAGs, and a TAGenriched product that is depleted in MAGs and/or DAGs. According to thefractionating conditions employed, in one embodiment of the presentmethods, the refined lipid composition may be depleted in FFAs relativeto the oil composition of the pelletized microbial biomass. In oneembodiment, the refined lipid composition may be enriched in at leastone PUFA relative to the oil composition of the pelletized microbialbiomass. In one embodiment, the refined lipid composition may beenriched in at least one PUFA having 20 or more carbon atoms relative tothe oil composition of the pelletized microbial biomass.

The fractionation may be performed by altering the temperature, thepressure, or the temperature and the pressure of the fractionatingconditions. Fractionation may be accomplished in one of severalseparation processes including, for example, a sequential pressurereduction of the supercritical fluid-rich extract, liquid or SCF solventextraction in a series of mixer-settler stages or extraction column,short-path distillation, vacuum steam stripping, or meltcrystallization. The step of fractionating the extract may be repeatedone or more times to provide additional refined lipid compositions.

Reducing the pressure, for example, of the extract lowers the solubilityof the dissolved solutes, forming a separate liquid phase in eachseparation vessel. The temperature of the extract being fed to eachseparation vessel can be adjusted, for example through the use of heatexchangers, to provide the desired solvent density and correspondingsolute solubility in each separation vessel. The initial extractconsists of a complex mixture of various types of lipid components(e.g., FFAs, MAGs, DAGs, and TAGs) which exhibit similar solubilityparameters, so an exact separation of the various components will not beachieved, but rather each refined lipid composition obtained in thefractionation step will contain a distribution of products. However, ingeneral, the less soluble compounds condense in the first separationvessel operating at the highest pressure, and the most soluble compoundscondense in the final separation vessel operating at the lowestpressure. The final separation vessel reduces the pressure of theextract phase sufficiently to essentially remove the bulk of theremaining solute in the extract phase, and the relatively pure CO₂stream from the top of this vessel may be recycled back to the initialextraction vessel(s).

FIG. 2 schematically illustrates one embodiment of the extractionmethods herein. In FIG. 2, stream 10 comprising pelletized microbialbiomass and stream 38 comprising CO₂ are shown entering vessel 14.Stream 12 comprising pelletized microbial biomass and stream 16 areshown entering vessel 18. Contacting of pelletized microbial biomasscomprising at least one PUFA with CO₂ occurs in vessel 14 at an initialtemperature T₁₄ and pressure P₁₄, and in vessel 18 at a temperature T₁₈and pressure P₁₈. T₁₄ may be the same as or different from T₁₈; P₁₄ maybe the same as or different from P₁₈. The resulting mixture ofequilibrated CO₂ and extract leaves vessel 14 as stream 16 to entervessel 18, in which further contacting of the biomass and the CO₂ occursto provide an extract comprising a lipid fraction substantially free ofPLs, shown as stream 20. The residual biomass (not shown) remains invessels 14 and 16. Additional extraction vessels may be included in theprocess, if desired (not shown). Alternatively, the process may use onlyone extraction vessel if desired (not shown). The use of more than oneextraction vessel may be advantageous as this can enable continuous CO₂flow through the process by changing the relative order of solventaddition to the extraction vessels (not shown) and while one or moreextraction vessels are taken off line (not shown), for example to chargepelletized microbial biomass or to remove residual biomass.

Downstream of the extraction vessels are shown two separation vesselsarranged in series, vessels 22 and 28, in which fractionation of theextract is performed through a staged pressure reduction, optionallywith adjustment of the temperature, for example through the use of heatexchangers (not shown). Additional separation vessels could be includedin the process, if desired (not shown). The extract comprising CO₂ and alipid fraction substantially free of PLs is shown entering vessel 22 asstream 20. In vessel 22, the pressure P₂₂ is lower than P₁₈ and thetemperature T₂₂ may be the same as or different from T₁₈; under theoperating conditions of the process, a separate liquid phase comprisingthe less soluble lipid components is formed. The separate liquid phaseresulting from fractionation of the extract is shown leaving vessel 22as stream 24, which represents a first refined lipid composition. Theremaining extract, shown as stream 26, is introduced to the nextseparation vessel 28, where the pressure P₂₈ is reduced compared to P₂₂and the temperature T₂₈ may or may not be the same as T₂₂. The operatingconditions of the process enable formation of a separate liquid phase invessel 28, which is shown leaving separation vessel 28 as stream 30.Stream 30 represents a second refined lipid composition.

From vessel 28, the remaining extract comprising relatively pure CO₂,shown as stream 32, may be recycled to extraction vessel 14 and/or toanother extraction vessel (not shown). Recycling the CO₂ typicallyprovides economic benefits over once-through CO₂ usage. A purge stream,shown as stream 34, can be used to remove volatile components which maybuild up with continuous recycle of the CO₂ to the process. Make-up CO₂may be added to offset the CO₂ loss incurred through a purge. Make-upCO₂ may be added to the recycle CO₂ stream as shown in FIG. 2 by make-upCO₂ stream 8 joining stream 36 to provide the combined CO₂ stream 38.Alternatively, additional CO₂ could be added to vessel 14 and/or vessel18 as a separate feed stream (not shown).

FIG. 3 schematically illustrates one embodiment of the extraction stepof the method of the invention. In FIG. 3, stream 70 comprising CO₂ isintroduced into extraction vessel 76, which contains pelletizedmicrobial biomass (not shown). Optionally, a cosolvent (shown as stream72) is added to the CO₂ stream using a pump (not shown) to provide thecombined stream 74 comprising CO₂ and cosolvent. In the case where acosolvent is not used, stream 70 and stream 74 are the same and containonly CO₂. Contacting the CO₂ with the pelletized microbial biomasscomprising at least one PUFA occurs in vessel 76, and the extractcomprising a lipid fraction substantially free of PLs is removed fromthe vessel as stream 78 along with the CO₂ solvent and optionally thecosolvent. The residual biomass (not shown) remains in the extractionvessel. The extract comprising a lipid fraction substantially free ofPLs may then be fractionated in at least one separation vessel, asdescribed above in reference to FIG. 2, or optionally, the lipidfraction substantially free of PLs may be isolated from the extract byventing the CO₂ and optionally the cosolvent (not shown).

The residual biomass from the above primary extraction comprises PLs.This residual biomass may be extracted a second time with a polarextraction solvent, for example a polar organic solvent such asmethylene chloride or a mixed solvent comprising CO₂ and a polarcosolvent such as an alcohol, to obtain a PL fraction free of neutrallipids. The polar cosolvent may comprise methanol, ethanol, 1-propanol,and/or 2-propanol, for example. The residual biomass comprising PLs andthe extracted PL fraction may be suitable for use as, e.g., anaquaculture feed.

The CO₂-based extraction process described herein offers severaladvantages relative to conventional organic solvent-based processes. Forexample, CO₂ is nontoxic, nonflammable, environmentally friendly,readily available, and inexpensive. CO₂ (T_(c)=31.1° C.) can extractthermally labile lipids from microbial biomass at relatively lowtemperatures to minimize lipid degradation in the oil. The extractedlipids may be isolated from the CO₂ solvent by simply venting the CO₂from the pressurized extract rather than through thermal processing tostrip organic solvents. The refined lipid fraction may be isolated fromthe extract comprising a lipid fraction substantially free of PLs. Theresidual microbial biomass containing PLs may be a saleable co-product,for example, for aquaculture feed. The PLs may be extracted from theresidual microbial biomass as a relatively pure co-product depleted inneutral lipids. The extracted neutral lipid fraction substantially freeof PLs may be fractionated to produce a lipid fraction enriched in FFAsand DAGs (and depleted in TAGs) relative to the lipid fractionsubstantially free of PLs and a refined lipid fraction enriched in TAGs(and depleted in FFAs and DAGs) relative to the lipid fractionsubstantially free of PLs.

The distillation step includes at least one pass of the extractedmicrobial oil (e.g., the refined lipid composition) through a short pathdistillation (SPD) still. Commercial SPD stills are well known in theart of chemical engineering. Suitable stills are available, for example,from Pope Scientific (Saukville, Wis.). The SPD still includes anevaporator and a condenser. A typical distillation is controlled by thetemperature of the evaporator, the temperature of the condenser, thefeed-rate of the oil into the still and the vacuum level of the still.

As one of skill in the art will appreciate, the number of passes througha SPD still will depend on the level of moisture in the extracted oil.If the moisture content is low, a single pass through the SPD still maybe sufficient.

Preferably, however, the distillation is a multi-pass process includingtwo or more consecutive passes of the extracted oil (e.g., the refinedlipid composition) through a SPD still. A first pass is typicallyperformed under about 1 to 50 torr pressure, and preferably about 5 to30 torr, with relatively low surface temperature of the evaporator, forinstance, about 100 to 150° C. This results in a dewatered oil, asresidual water and low molecular weight organic materials are distilled.The dewatered oil is then passed through the still at higher temperatureof the evaporator and lower pressures to provide a distillate fractionand a TAG-containing fraction (i.e., the lipid-containing fraction).

In some embodiments, the extracted oil comprises a sterol fraction,which may removed following distillation under SPD conditions. Morespecifically, when the extracted oil comprises a sterol fraction,distillation at least once under short path distillation conditionsresults in a distillate fraction comprising the sterol and alipid-containing fraction comprising a reduced amount of the sterol whencompared to the amount of the sterol in the extracted oil that has notbeen subjected to short path distillation. As previously discussed, thesterol fraction may comprise one or more sterols selected from the groupconsisting of: stigmasterol, ergosterol, brassicasterol, campesterol,β-sitosterol and desmosterol.

Additional passes of the TAG-containing lipid fraction may be madethrough the still to remove further sterol. With each additional pass,the distillation temperature may be increased relative to thetemperature of the immediately preceding distillation. Preferably,sufficient passes are performed such that the reduction in the amount ofthe sterol fraction is at least about 40%-70%, preferably at least about70%-80%, and more preferably greater than about 80%, when compared tothe sterol fraction in the sterol-containing microbial oil.

Preferably, the SPD conditions comprise at least one pass of thesterol-containing microbial oil (i.e., the refined lipid composition) ata vacuum level of not more than 30 mTorr, and preferably not more than 5mTorr. Preferably, the SPD conditions comprise at least one pass atabout 220 to 300° C., and preferably at about 240 to 280° C.

Thus, for example, in one embodiment, the extracted oil is a refinedlipid composition comprising: (i) at least one PUFA and enriched in TAGs(relative to the oil composition of pelletized microbial biomass that isnot processed with a solvent); and (ii) a sterol fraction of at least300 mg/100 g. When subjected to distillation at least once under SPDconditions, the distillation produces a distillate fraction comprisingthe sterol and a lipid-containing fraction comprising TAGs having areduced sterol fraction that has improved clarity when compared to therefined lipid composition that has not been subjected to SPD. Improvedclarity refers to a lack of cloudiness or opaqueness in the oil.Sterol-containing microbial oil becomes cloudy upon storing attemperatures below about 10° C., due to reduced solubility of the sterolin the oil at lower temperatures. The distillation process acts toremove substantial portions of the sterol fraction, such that theresulting lipid-containing fraction has a reduced amount of sterolpresent, and thus, remains clear, or substantially clear upon storage atabout 10° C. A test method that may be used to evaluate the clarity ofthe oil is the American Oil Chemists' Society (AOCS) Official Method Cc11-53 (“Cold Test”, Official Methods and Recommended Practices of theAOCS, 6^(th) ed., Urbana, Ill., AOCS, 2009, incorporated herein byreference).

Surprisingly, the removal of sterol in the distillation process can beaccomplished without significant degradation of the oil, which is richin PUFAs, e.g., EPA. The degradation of the oil may be evaluated basedon the PUFA content and chromatographic profiling (as demonstrated inExample 23, infra).

Recovering the lipid-containing fraction may be accomplished bydiverting the fraction, after completion of a pass through theevaporator, to a suitable container.

The fatty acids in an extracted microbial oil or product thereof (e.g.,a lipid-containing fraction) are typically in a biological form such asa triglyceride or phospholipid. Because it is difficult to enrich thefatty acid profile of these forms, the individual fatty acids of themicrobial oil will usually be liberated by transesterification usingtechniques well known to those skilled in the art. Since the fatty acidester mixture has substantially the same fatty acid profile as themicrobial oil prior to transesterification, the product of thetransesterification process is still typically considered anon-concentrated microbial oil (i.e., in ester form).

Enrichment of a microbial oil comprising 30 to 70 wt % of a desiredPUFA, measured as a wt % of TFAs (wherein the microbial oil is obtainedfrom an oil-containing microbe that accumulates in excess of 25% of itsdry cell weight as oil) results in an oil concentrate which comprises atleast 70 wt % of the desired PUFA, measured as a wt % of oil (i.e., an“oil concentrate”). Specifically, the ethyl or other esters of themicrobial oil can be enriched in the desired PUFA (e.g., LA, EDA, GLA,DGLA, ARA, DTA, DPAn-6, ALA, STA, ETrA, ETA, EPA, DPAn-3, DHA) andseparated by methods commonly used in the art, such as: fractionaldistillation, urea adduct formation, short path distillation,supercritical fluid fractionation with counter current column,supercritical fluid chromatography, liquid chromatography, enzymaticseparation and treatment with silver salt, simulated moving bedchromatography, actual moving bed chromatography and combinationsthereof.

Thus, for example, disclosed herein is a method for making an EPAconcentrate comprising at least 70 wt % EPA, measured as a wt % of oiland substantially free of DHA, said method comprising:

-   -   a) transesterifying the lipid-containing fraction of the present        invention comprising 30 to 70 wt % EPA, measured as a wt % of        TFAs, and substantially free of DHA; and,    -   b) enriching the transesterified oil of step (a) to obtain an        EPA concentrate comprising at least 70 wt % EPA, measured as a        wt % of oil, and substantially free of DHA.

For example, a non-concentrated purified microbial oil (i.e., thelipid-containing fraction) comprising 58.2% EPA, measured as a wt % ofTFAs, and substantially free of DHA from Yarrowia lipolytica is providedin Example 27 herein. This lipid-containing fraction is enriched inExample 28 via a urea adduct formation method, such that the resultingEPA ethyl ester (EPA-EE) concentrate comprises 76.5% EPA-EE, measured asa wt % of oil, and is substantially free of DHA. Similarly, Example 29demonstrates enrichment of the same lipid-containing fraction via liquidchromatography, wherein the resulting EPA-EE concentrate comprises 82.8%or 95.4% EPA-EE, measured as a wt % of oil, and is substantially free ofDHA. Example 30 demonstrates enrichment of the same lipid-containingfraction via supercritical fluid chromatography, resulting in an EPAconcentrate comprising 85% or 89.8% EPA-EE, measured as a wt % of oil,that is substantially free of DHA.

An alternate non-concentrated SPD-purified microbial oil (i.e., thelipid-containing fraction) comprising 56.1% EPA, measured as a wt % ofTFAs, and substantially free of DHA from Yarrowia lipolytica is providedin Example 31. Enrichment of this lipid-containing fraction in Example32 occurs via fractional distillation, thereby producing an EPAconcentrate that comprises 73% EPA-EE, measured as a wt % of oil, and issubstantially free of DHA. Fractional distillation advantageouslyremoves many of the lower molecular weight ethyl esters present in theoil (i.e., predominantly C18s in the lipid-containing fraction ofExample 32, but not limited thereto).

An alternate non-concentrated SPD-purified microbial oil (i.e., thelipid-containing fraction) comprising 54.7% EPA, measured as a wt % ofTFAs, and substantially free of DHA, NDPA and HPA from Yarrowialipolytica is provided in Example 34. Enrichment of thislipid-containing fraction occurs via fractional distillation and liquidchromatography, thereby producing an EPA concentrate that comprises97.4% EPA-EE, measured as a wt % of oil, and is substantially free ofDHA, NDPA and HPA. One of skill in the art should appreciate that othercombinations of enrichment processes (e.g., fractional distillation,urea adduct formation, short path distillation, supercritical fluidfractionation with counter current column, supercritical fluidchromatography, liquid chromatography, enzymatic separation andtreatment with silver salt, simulated moving bed chromatography, actualmoving bed chromatography) could be utilized to produce an EPAconcentrate of the present invention.

For example, it may be particularly advantageous to make an EPAconcentrate comprising at least 70 wt % of EPA, measured as a wt % ofoil, and substantially free of DHA, said method comprising: (a) atransesterification reaction of a lipid-containing fraction comprising30 to 70 wt % of EPA, measured as a wt % of TFAs; (b) a first enrichmentprocess comprising fractional distillation for removal of many of thelower molecular weight ethyl esters, i.e., comprising C14, C16 and C18fatty acids; and, (c) at least one additional enrichment processselected from the group consisting of: urea adduct formation, liquidchromatography, supercritical fluid chromatography, simulated moving bedchromatography, actual moving bed chromatography and combinationsthereof. Lower concentrations of C14, C16 and C18 fatty acids in the oilsample, as a result of fractional distillation, may facilitatesubsequent enrichment processes.

As will be recognized by one of skill in the art, any of the EPAconcentrates described above, in ethyl ester form, can readily beconverted, if desired, to other forms such as, for example, a methylester, an acid or a triacylglyceride, or any other suitable form or acombination thereof. Means for chemical conversion of PUFAs from onederivative to another is well known. For example, triglycerides can beconverted to sodium salts of the cleaved acids by saponification andfurther to free fatty acids by acidification, and ethyl esters can bere-esterified to triglycerides via glycerolysis. Thus, while it isexpected that the EPA concentrate will initially be in the form of anethyl ester, this is by no means intended as a limitation. The at least70 wt % EPA, measured as a wt % of oil, within an EPA concentrate willtherefore refer to EPA in the form of free fatty acids,triacylglycerols, esters, and combinations thereof, wherein the estersare most preferably in the form of ethyl esters.

One of ordinary skill in the art will appreciate that processingconditions can be optimized to result in any preferred level of PUFAenrichment of the lipid-containing fraction, such that the desired PUFAconcentrate has at least 70 wt % desired PUFA, measured as a wt % of oil(although increased PUFA purity is often inversely related to PUFAyield). Thus, those skilled in the art will appreciate that the wt % ofa desired PUFA can be any integer percentage (or fraction thereof) from70% up to and including 100%, i.e., specifically, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% thedesired PUFA, measured as a wt % of oil.

More specifically, in one embodiment of the present invention, there isprovided an EPA concentrate comprising at least 80 wt % of EPA, measuredas a wt % of oil, and substantially free of DHA. In another embodiment,there is provided an EPA concentrate comprising at least 90 wt % of EPA,measured as a wt % of oil, and substantially free of DHA. And, in yetanother embodiment, there is provided an EPA concentrate comprising atleast 95 wt % of EPA, measured as a wt % of oil, and substantially freeof DHA.

In preferred embodiments, the EPA concentrates described above,comprising at least 70 wt % EPA, measured as a wt % of oil, andsubstantially free of DHA can be further characterized as substantiallyfree of NDPA and substantially free of HPA.

Although not limited to any particular application, the PUFAconcentrates of the present invention are particularly well suited foruse as a pharmaceutical. As is well known to one of skill in the art,PUFAs may be administered in a capsule, a tablet, a granule, a powderthat can be dispersed in a beverage, or another solid oral dosage form,a liquid (e.g., syrup), a soft gel capsule, a coated soft gel capsule orother convenient dosage form such as oral liquid in a capsule. Capsulesmay be hard-shelled or soft-shelled and may be of a gelatin orvegetarian source. PUFAs may also be contained in a liquid suitable forinjection or infusion.

Additionally, PUFAs may also be administered with a combination of oneor more non-active pharmaceutical ingredients (also known generallyherein as “excipients”). Non-active ingredients, for example, serve tosolubilize, suspend, thicken, dilute, emulsify, stabilize, preserve,protect, color, flavor, and fashion the active ingredients into anapplicable and efficacious preparation that is safe, convenient, andotherwise acceptable for use.

Excipients may include, but are not limited to, surfactants, such aspropylene glycol monocaprylate, mixtures of glycerol and polyethyleneglycol esters of long fatty acids, polyethoxylated castor oils, glycerolesters, oleoyl macrogol glycerides, propylene glycol monolaurate,propylene glycol dicaprylate/dicaprate, polyethylene-polypropyleneglycol copolymer and polyoxyethylene sorbitan monooleate, cosolventssuch as ethanol, glycerol, polyethylene glycol, and propylene glycol,and oils such as coconut, olive or safflower oils. The use ofsurfactants, cosolvents, oils or combinations thereof is generally knownin the pharmaceutical arts, and as would be understood to one skilled inthe art, any suitable surfactant may be used in conjunction with thepresent invention and embodiments thereof.

The dose concentration, dose schedule and period of administration ofthe composition should be sufficient for the expression of the intendedaction, and may be adequately adjusted depending on, for example, thedosage form, administration route, severity of the symptom(s), bodyweight, age and the like. When orally administered, the composition maybe administered in three divided doses per day, although the compositionmay alternatively be administered in a single dose or in several divideddoses.

Extracted oil compositions comprising at least one PUFA, such as EPA (orderivatives thereof), will have well known clinical and pharmaceuticalvalue. See, e.g., U.S. Pat. Appl. Pub. No. 2009-0093543 A1. For example,lipid compositions comprising PUFAs may be used as dietary substitutes,or supplements, particularly infant formulas, for patients undergoingintravenous feeding or for preventing or treating malnutrition.Alternatively, the purified PUFAs (or derivatives thereof) may beincorporated into cooking oils, fats or margarines formulated so that innormal use the recipient would receive the desired amount for dietarysupplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use, either human orveterinary.

Supplementation of humans or animals with PUFAs can result in increasedlevels of the added PUFAs, as well as their metabolic progeny. Forexample, treatment with EPA can result not only in increased levels ofEPA, but also downstream products of EPA such as eicosanoids (i.e.,prostaglandins, leukotrienes, thromboxanes), DPAn-3 and DHA. Complexregulatory mechanisms can make it desirable to combine various PUFAs, oradd different conjugates of PUFAs, in order to prevent, control orovercome such mechanisms to achieve the desired levels of specific PUFAsin an individual.

Alternatively, PUFAs, or derivatives thereof, can be utilized in thesynthesis of animal and aquaculture feeds, such as dry feeds, semi-moistand wet feeds, since these formulations generally require at least 1-2%of the nutrient composition to be omega-3 and/or omega-6 PUFAs.

EXAMPLES

The present invention is further defined in the following examples. Itshould be understood that these examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The following abbreviations are used: “HPLC” is High Performance LiquidChromatography, “ASTM” is American Society for Testing And Materials,“C” is Celsius, “kPa” is kiloPascal, “mm” is millimeter, “μm” ismicrometer, “μL” is microliter, “mL” is milliliter, “L” is liter, “min”is minute, “mM” is millimolar, “mTorr” is milliTorr, “cm” is centimeter,“g” is gram, “wt” is weight, “h” or “hr” is hour, “temp” or “T” istemperature, “SS” is stainless steel, “in” is inch, “i.d.” is insidediameter, and “o.d.” is outside diameter.

Materials Biomass Preparation

Described below are strains of Yarrowia lipolytica yeast producingvarious amounts of microbial oil comprising PUFAs. Biomass was obtainedin a 2-stage fed-batch fermentation process, and then subjected todownstream processing, as described below.

Yarrowia lipolytica Strains: The generation of Yarrowia lipolyticastrain Y8672 is described in U.S. Pat. Appl. Pub. No. 2010-0317072-A1.Strain Y8672, derived from Y. lipolytica ATCC #20362, was capable ofproducing about 61.8% EPA relative to the total lipids via expression ofa delta-9 elongase/delta-8 desaturase pathway.

The final genotype of strain Y8672 with respect to wild type Yarrowialipolytica ATCC #20362 was Ura+, Pex3−, unknown 1−, unknown 2−, unknown3−, unknown 4−, unknown 5−, unknown 6−, unknown 7−, unknown 8−, Leu+,Lys+, YAT1::ME3S::Pex16, GPD::ME3S::Pex20, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::FmD12S::ACO, GPAT::EgD9e::Lip2,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, YAT1::EgD9eS::Lip2,FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1, EXP1::EgD8M::Pex16,GPD::EaD8S::Pex16 (2 copies), YAT1::E389D9eS/EgD8M::Lip1,YAT1::EgD9eS/EgD8M::Aco, FBAIN::EgD5SM::Pex20, YAT1::EgD5SM::Aco,GPM::EgD5SM::Oct, EXP1::EgD5M::Pex16, EXP1::EgD5SM::Lip1,YAT1::EaD5SM::Oct, YAT1::PaD17S::Lip1, EXP1::PaD17::Pex16,FBAINm::PaD17::Aco, GPD::YICPT1::Aco, and YAT1::MCS::Lip1.

The structure of the above expression cassettes are represented by asimple notation system of “X::Y::Z”, wherein X describes the promoterfragment, Y describes the gene fragment, and Z describes the terminatorfragment, which are all operably linked to one another. Abbreviationsare as follows: FmD12 is a Fusarium moniliforme delta-12 desaturase gene[U.S. Pat. No. 7,504,259]; FmD12S is a codon-optimized delta-12desaturase gene, derived from F. moniliforme [U.S. Pat. No. 7,504,259];MESS is a codon-optimized C_(16/18) elongase gene, derived fromMortierella alpina [U.S. Pat. No. 7,470,532]; EgD9e is a Euglenagracilis delta-9 elongase gene [U.S. Pat. No. 7,645,604]; EgD9eS is acodon-optimized delta-9 elongase gene, derived from E. gracilis [U.S.Pat. No. 7,645,604]; EgD8M is a synthetic mutant delta-8 desaturase gene[U.S. Pat. No. 7,709,239], derived from E. gracilis [U.S. Pat. No.7,256,033]; EaD8S is a codon-optimized delta-8 desaturase gene, derivedfrom Euglena anabaena [U.S. Pat. No. 7,790,156]; E389D9eS/EgD8M is aDGLA synthase created by linking a codon-optimized delta-9 elongase gene(“E389D9eS”), derived from Eutreptiella sp. CCMP389 delta-9 elongase(U.S. Pat. No. 7,645,604) to the delta-8 desaturase “EgD8M” (supra)[U.S. Pat. Appl. Pub. No. 2008-0254191-A1]; EgD9ES/EgD8M is a DGLAsynthase created by linking the delta-9 elongase “EgD9eS” (supra) to thedelta-8 desaturase “EgD8M” (supra) [U.S. Pat. Appl. Pub. No.2008-0254191-A1]; EgD5M and EgD5SM are synthetic mutant delta-5desaturase genes [U.S. Pat. App. Pub. 2010-0075386-A1], derived from E.gracilis [U.S. Pat. No. 7,678,560]; EaD5SM is a synthetic mutant delta-5desaturase gene [U.S. Pat. App. Pub. 2010-0075386-A1], derived from E.anabaena [U.S. Pat. No. 7,943,365]; PaD17 is a Pythium aphanidermatumdelta-17 desaturase gene [U.S. Pat. No. 7,556,949]; PaD17S is acodon-optimized delta-17 desaturase gene, derived from P. aphanidermatum[U.S. Pat. No. 7,556,949]; YICPT1 is a Y. lipolytica diacylglycerolcholinephosphotransferase gene [U.S. Pat. No. 7,932,077]; and, MCS is acodon-optimized malonyl-CoA synthetase gene, derived from Rhizobiumleguminosarum bv. viciae 3841 [U.S. Pat. App. Pub. 2010-0159558-A1].

For a detailed analysis of the total lipid content and composition instrain Y8672, a flask assay was conducted wherein cells were grown in 2stages for a total of 7 days. Based on analyses, strain Y8672 produced3.3 g/L dry cell weight [“DCW”], total lipid content of the cells was26.5 [“TFAs % DCW”], the EPA content as a percent of the dry cell weight[“EPA % DCW”] was 16.4, and the lipid profile was as follows, whereinthe concentration of each fatty acid is as a weight percent of TFAs [“%TFAs”]: 16:0 (palmitate)—2.3, 16:1 (palmitoleic acid)—0.4, 18:0 (stearicacid)—2.0, 18:1 (oleic acid)—4.0, 18:2 (LA)—16.1, ALA—1.4, EDA—1.8,DGLA—1.6, ARA—0.7, ETrA—0.4, ETA—1.1, EPA—61.8, other—6.4.

The generation of Yarrowia lipolytica strain Y9502 is described in U.S.Pat. Appl. Pub. No. 2010-0317072-A1, hereby incorporated herein byreference in its entirety. Strain Y9502, derived from Y. lipolytica ATCC#20362, was capable of producing about 57.0% EPA relative to the totallipids via expression of a delta-9 elongase/delta-8 desaturase pathway.

The final genotype of strain Y9502 with respect to wildtype Y.lipolytica ATCC #20362 was Ura+, Pex3−, unknown 1−, unknown 2−, unknown3−, unknown 4−, unknown 5−, unknown6−, unknown 7−, unknown 8−,unknown9−, unknown 10−, YAT1::ME3S::Pex16, GPD::ME3S::Pex20,YAT1::ME3S::Lip1, FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1,GPAT::EgD9e::Lip2, YAT1::EgD9eS::Lip2, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::EaD8S::Pex16 (2 copies),YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAINm::EaD9eS/EaD8S::Lip2, GPD::FmD12::Pex20, YAT1::FmD12::Oct,EXP1::FmD12S::Aco, GPDIN::FmD12::Pex16, EXP1::EgD5M::Pex16,FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco, GPM::EgD5SM::Oct,EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct, FBAINm::PaD17::Aco,EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1, YAT1::YICPT::Aco,YAT1::MCS::Lip1, FBA::MCS::Lip1, YAT1::MaLPAAT1S::Pex16.

Abbreviations not previously defined are as follows: EaD9eS/EgD8M is aDGLA synthase created by linking a codon-optimized delta-9 elongase gene(“EaD9eS”), derived from Euglena anabaena delta-9 elongase [U.S. Pat.No. 7,794,701] to the delta-8 desaturase “EgD8M” (supra) [U.S. Pat.Appl. Pub. No. 2008-0254191-A1]; and, MaLPAAT1S is a codon-optimizedlysophosphatidic acid acyltransferase gene, derived from Mortierellaalpina [U.S. Pat. No. 7,879,591].

For a detailed analysis of the total lipid content and composition instrain Y9502, a flask assay was conducted wherein cells were grown in 2stages for a total of 7 days. Based on analyses, strain Y9502 produced3.8 g/L dry cell weight [“DCW”], total lipid content of the cells was37.1 [“TFAs % DCW”], the EPA content as a percent of the dry cell weight[“EPA % DCW”] was 21.3, and the lipid profile was as follows, whereinthe concentration of each fatty acid is as a weight percent of TFAs [“%TFAs”]: 16:0 (palmitate)—2.5, 16:1 (palmitoleic acid)—0.5, 18:0 (stearicacid)—2.9, 18:1 (oleic acid)—5.0, 18:2 (LA)—12.7, ALA—0.9, EDA—3.5,DGLA—3.3, ARA—0.8, ETrA—0.7, ETA—2.4, EPA—57.0, other—7.5.

The generation of Yarrowia lipolytica strain Y4305F1B1, derived from Y.lipolytica ATCC #20362 and capable of producing about 50-52% EPArelative to the total lipids with 28-32% total lipid content [“TFAsDCW”] via expression of a delta-9 elongase/delta-8 desaturase pathway,is described in U.S. Pat. Appl. Pub. No. 2011-0059204-A1, herebyincorporated herein by reference in its entirety. Specifically, strainY4305F1B1 is derived from Y. lipolytica strain Y4305, which has beenpreviously described in the General Methods of U.S. Pat. App. Pub. No.2008-0254191, the disclosure of which is hereby incorporated in itsentirety.

The final genotype of strain Y4305 with respect to wild type Y.lipolytica ATCC #20362 was SCP2− (YALI0E01298g), YALI0C18711g−, Pex10−,YALI0F24167g−, unknown 1−, unknown 3−, unknown 8−, GPD::FmD12::Pex20,YAT1::FmD12::OCT, GPM/FBAIN::FmD12S::OCT, EXP1::FmD12S::Aco,YAT1::FmD12S::Lip2, YAT1::ME3S::Pex16, EXP1::ME3S::Pex20 (3 copies),GPAT::EgD9e::Lip2, EXP1::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2,FBA::EgD9eS::Pex20, GPD::EgD9eS::Lip2, YAT1::EgD9eS::Lip2,YAT1::E389D9eS::OCT, FBAINm::EgD8M::Pex20, FBAIN::EgD8M::Lip1 (2copies), EXP1::EgD8M::Pex16, GPDIN::EgD8M::Lip1, YAT1::EgD8M::Aco,FBAIN::EgD5::Aco, EXP1::EgD5S::Pex20, YAT1::EgD5S::Aco,EXP1::EgD5S::ACO, YAT1::RD5S::OCT, YAT1::PaD17S::Lip1,EXP1::PaD17::Pex16, FBAINm::PaD17::Aco, YAT1::YICPT1::ACO,GPD::YICPT1::ACO.

Abbreviations not previously defined are as follows: EgD5 is a Euglenagracilis delta-5 desaturase [U.S. Pat. No. 7,678,560]; EgD5S is acodon-optimized delta-5 desaturase gene, derived from E. gracilis [U.S.Pat. No. 7,678,560]; and, RD5S is a codon-optimized delta-5 desaturase,derived from Peridinium sp. CCMP626 [U.S. Pat. No. 7,695,950].

Total lipid content of the Y4305 cells was 27.5 [“TFAs % DCW”], and thelipid profile was as follows, wherein the concentration of each fattyacid is as a weight percent of TFAs [“% TFAs”]: 16:0 (palmitate)—2.8,16:1 (palmitoleic acid)—0.7, 18:0 (stearic acid)—1.3, 18:1 (oleicacid)—4.9, 18:2 (LA)—17.6, ALA—2.3, EDA—3.4, DGLA—2.0, ARA—0.6, ETA—1.7and EPA—53.2.

Strain Y4305 was subjected to transformation with a dominant,non-antibiotic marker for Y. lipolytica based on sulfonylurea [“SU^(R)”]resistance. More specifically, the marker gene is a nativeacetohydroxyacid synthase (“AHAS” or acetolactate synthase; E.C.4.1.3.18) that has a single amino acid change, i.e., W497L, that conferssulfonyl urea herbicide resistance (SEQ ID NO:292 of Intl. App. Pub. No.WO 2006/052870). The random integration of the SU^(R) genetic markerinto Yarrowia strain Y4305 was used to identify those cells havingincreased lipid content when grown under oleaginous conditions relativeto the parent Y4305 strain, as described in U.S. Pat. App. Pub. No.2011-0059204-A1.

When evaluated under 2 liter fermentation conditions, average EPAproductivity [“EPA % DCW”] for strain Y4305 was 50-56, as compared to50-52 for mutant SU^(R) strain Y4305-F1B1. Average lipid content [“TFAsDCW”] for strain Y4305 was 20-25, as compared to 28-32 for strainY4305-F1B1. Thus, lipid content was increased 29-38% in strainY4503-F1B1, with minimal impact upon EPA productivity.

Fermentation: Inocula were prepared from frozen cultures of Y.lipolytica in a shake flask. After an incubation period, the culture wasused to inoculate a seed fermentor. When the seed culture reached anappropriate target cell density, it was then used to inoculate a largerfermentor. The fermentation is a 2-stage fed-batch process. In the firststage, the yeast were cultured under conditions that promote rapidgrowth to a high cell density; the culture medium comprised glucose,various nitrogen sources, trace metals and vitamins. In the secondstage, the yeast were starved for nitrogen and continuously fed glucoseto promote lipid and PUFA accumulation. Process variables includingtemperature (controlled between 30-32° C.), pH (controlled between 5-7),dissolved oxygen concentration and glucose concentration were monitoredand controlled per standard operating conditions to ensure consistentprocess performance and final PUFA oil quality.

One of skill in the art of fermentation will know that variability willoccur in the oil profile of a specific Yarrowia strain, depending on thefermentation run itself, media conditions, process parameters, scale-up,etc., as well as the particular time-point in which the culture issampled (see, e.g., U.S. Pat. Appl. Pub. No. 2009-0093543-A1).

Downstream Processing: Antioxidants were optionally added to thefermentation broth prior to processing to ensure the oxidative stabilityof the EPA oil. The yeast biomass was dewatered and washed to removesalts and residual medium, and to minimize lipase activity. Eitherdrum-drying (typically with 80 psig steam) or spray-drying was thenperformed, to reduce moisture level to less than 5% to ensure oilstability during short term storage and transportation. The drum driedflakes, or spray dried powder having particle size distribution in rangeof about 10 to 100 micron, were used in the following ComparativeExamples and Examples, as the initial “microbial biomass, comprisingoil-containing microbes”.

Grinding Agents: Celite 209 D-earth is available from CeliteCorporation, Lompoc, Calif. Celatom MN-4 D-earth is available from EPMinerals, An Eagle Pitcher Company, Reno, Nev.

Other Materials: All commercial reagents were used as received. Allsolvents used were HPLC grade. Acetyl chloride was 99+%. TLC plates andsolvents were obtained from VWR (West Chester, Pa.). HPLC or SCF gradecarbon dioxide was obtained from MG Industries (Malvern, Pa.).

Twin Screw Extrusion Method

Twin screw extrusion was used in disrupting dried yeast biomass andpreparing disrupted biomass mix with grinding agents.

Dried yeast is fed into an extruder, preferably a twin screw extruderwith a length, normally 21-39 L/D, suitable for accomplishing theoperations described below. The first section of the extruder is used tofeed and transport the materials. The second section is a compactionzone designed to compact and compress the feed using bushing elementswith progressively shorter pitch length. After the compaction zone, acompression zone follows which serves to impart most of the mechanicalenergy required for cell disruption. This zone is created using flowrestriction either in the form of reverse screw elements,restriction/blister ring elements or kneading elements. When preparingdisrupted biomass, the grinding agent (e.g., D-earth) is typicallyco-fed with the microbial biomass feed so that both go through thecompression/compaction zone, thus enhancing disruption levels. Followingthe compression zone, the binding agent (e.g., water/sucrose solution)is added through a liquid injection port and mixed in subsequent mixingsections comprised of various combinations of mixing elements. The finalmixture (i.e., the “fixable mix”) is discharged through the last barrelwhich is open at the end, thus producing little or no backpressure inthe extruder. The fixable mix is then fed into a dome granulator andeither a vibrating or a fluidized bed drier. This results in pelletizedmaterial (i.e., solid pellets) suitable for downstream oil extraction.

SCF Extraction With CO₂

Dried and mechanically disrupted yeast cells were generally charged toan extraction vessel packed between plugs of glass wool, flushed withCO₂, and then heated and pressurized to the desired operating conditionsunder CO₂ flow. The CO₂ was fed directly from a commercial cylinderequipped with an eductor tube and was metered with a high-pressure pump.Pressure was maintained on the extraction vessel through use of arestrictor on the effluent side of the vessel, and the oil sample wascollected in a sample vessel while simultaneously venting the CO₂solvent to the atmosphere. A cosolvent (e.g., ethanol) could optionallybe added to the extraction solvent fed to the extraction vessel throughuse of a cosolvent pump (Isco Model 100D syringe pump).

Unless otherwise noted, supercritical CO₂ extraction of yeast samples inthe examples below was conducted in a custom high-pressure extractionapparatus (FIG. 1). In general, dried and mechanically disrupted yeastcells (free flowing or pelletized) were charged to an extraction vessel(1) packed between plugs of glass wool, flushed with CO₂, and thenheated and pressurized to the desired operating conditions under CO₂flow. The 89-ml extraction vessels were fabricated from 316 SS tubing(2.54 cm o.d.×1.93 cm i.d.×30.5 cm long) and equipped with a 2-micronsintered metal filter on the effluent end of the vessel. The extractionvessel was installed inside of a custom machined aluminum block equippedwith four calrod heating cartridges which were controlled by anautomated temperature controller. The CO₂ was fed as a liquid directlyfrom a commercial cylinder (2) equipped with an eductor tube and wasmetered with a high-pressure positive displacement pump (3) equippedwith a refrigerated head assembly (Jasco Model PU-1580-002). Extractionpressure was maintained with an automated back pressure regulator (4)(Jasco Model BP-1580-81) which provided a flow restriction on theeffluent side of the vessel, and the extracted oil sample was collectedin a sample vessel while simultaneously venting the CO₂ solvent to theatmosphere.

Reported oil extraction yields from the yeast samples were determinedgravimetrically by measuring the mass loss from the sample during theextraction. Thus, the reported extracted oil comprises microbial oil andmoisture associated with the solid pellets.

EXAMPLES Comparative Examples C1, C2A, C2B, Example 1, Example 2 andComparative Examples C3 and C4 Comparison of Means to Create a DisruptedBiomass Mix from Drum-Dried Flakes of Yarrowia lipolytica

Comparative Examples C1, C2A, C2B, C3 and C4 and Examples 1 and 2describe a series of comparative tests performed to optimize disruptionof drum dried flakes of yeast (i.e., Yarrowia lipolytica strain Y8672).Specifically, hammer milling with and without the addition of grindingagent was examined, as well as use of either a single screw or twinscrew extruder. Results are compared based on the total free microbialoil and disruption efficiency of the microbial cells, as well as thetotal extraction yield (based on supercritical CO₂ extraction).

Comparative Example C1 Hammer-Milled Yeast Powder without Grinding Agent

Drum dried flakes of yeast (Y. lipolytica strain Y8672) biomasscontaining 24.2% total oil (dry weight) were hammer-milled (MikropulBantam mill at a feed rate of 12 Kg/h) at ambient temperature using ajump-gap separator at 16,000 rpm with three hammers to provide milledpowder. Particle size of the milled powder was d10=3 μm; d50=16 μm andd90=108 μm, analyzed suspended in water using Frauenhofer laserdiffraction.

Comparative Example C2A Hammer-Milled Yeast Powder with Grinding Agentand Air Mill Mixing

The hammer-milled yeast powder provided by Comparative Example C1 (833g) was mixed with Celite 209 diatomaceous earth (D-earth) (167 g) in anair (jet) mill (Fluid Energy Jet-o-mizer 0101, at a feed rate of 6 Kg/h)for about 20 min at ambient temperature.

Comparative Example C2B Hammer-Milled Yeast Powder with Grinding Agentand Manual Mixing

Hammer-milled yeast powder provided by Comparative Example C1 (833 g)was mixed manually with Celite 209 D-earth (167 g) in a plastic bag.

Example 1 Hammer Milled Yeast Powder with Grinding Agent, Manual Mixing,and Single Screw Extruder

The hammer-milled yeast powder with D-earth from Comparative Example C2B(1000 g) was mixed with a 17.6 wt % aqueous sucrose solution (62.5 gsucrose in 291.6 g water) in a Hobart mixer for about 2.5 min and thenextruded (50-200 psi, torque not exceeding 550 in-lbs; 40° C. or lessextrudate temperature) through a single screw dome granulator having 1mm orifices. The extrudate was dried in a fluid bed dryer to a bedtemperature of 50° C. using fluidizing air controlled at 65° C. toprovide non-sticky pellets (815 g, having dimensions of 2 to 8 mm lengthand about 1 mm diameter) having 3.9% water remaining after about 14 min.

Example 2 Hammer Milled Yeast Powder with Grinding Agent, Air MillMixing, and Single Screw Extruder

The hammer milled yeast powder with D-earth from Comparative Example C2A(1000 g) was processed according to Example 1 to provide pellets (855 g,having dimensions of 2 to 8 mm length and about 1 mm diameter) having6.9% water remaining after about 10 min.

Comparative Example C3 Hammer Milled Yeast Powder without Grinding Agentand with Twin Screw Extruder

The hammer milled yeast powder provided from Comparative Example C1 wasfed at 2.3 kg/hr to an 18 mm twin screw extruder (Coperion WernerPfleiderer ZSK-18 mm MC, Stuttgart, Germany) operating with a 10 kWmotor and high torque shaft, at 150 rpm and % torque range of 66-68 toprovide a disrupted yeast powder cooled to 26° C. in a final watercooled barrel.

Comparative Example C4 Yeast Powder without Grinding Agent and with TwinScrew Extruder

Drum dried flakes of yeast (Y. lipolytica strain Y8672) biomasscontaining 24.2% total oil were fed at 2.3 kg/hr to an 18 mm twin screwextruder (Coperion Werner Pfleiderer ZSK-18 mm MC) operating with a 10kW motor and high torque shaft, at 150 rpm and % torque range of 71-73to provide a disrupted yeast powder cooled to 23° C. in a final watercooled barrel.

Comparison of Free Microbial Oil and Disruption Efficiency in DisruptedYeast Powder

The free microbial oil and disruption efficiency was determined in thedisrupted yeast powders of Examples 1 and 2, and Comparative ExamplesC1-C4 according to the following method. Specifically, free oil andtotal oil content of extruded biomass samples were determined using amodified version of the method reported by Troeng (J. Amer. Oil ChemistsSoc., 32:124-126 (1955)). In this method, a sample of the extrudedbiomass was weighed into a stainless steel centrifuge tube with ameasured volume of hexane. Several chrome steel ball bearings were addedif total oil was to be determined. The ball bearings were not used iffree oil was to be determined. The tubes were then capped and placed ona shaker for 2 hr. The shaken samples were centrifuged, the supernatantwas collected and the volume measured. The hexane was evaporated fromthe supernatant first by rotary film evaporation and then by evaporationunder a stream of dry nitrogen until a constant weight was obtained.This weight was then used to calculate the percentage of free or totaloil in the original sample. The oil content is expressed on a percentdry weight basis by measuring the moisture content of the sample, andcorrecting as appropriate.

The percent disruption efficiency (i.e., the percent of cells walls thathave been fractured during processing) was quantified by opticalvisualization.

Table 7 summarizes the yeast cell disruption efficiency data forExamples 1 and 2, and Comparative Examples C1-C4, and reveals thefollowing:

Comparative Example C1 shows that Hammer milling in the absence ofgrinding agent results in 33% disruption of the yeast cells.

Comparative Example C2A shows that air jet milling of Hammer-milledyeast in the presence of grinding agent increases the disruption of theyeast cells to 62%.

Example 1 shows that further mixing of Hammer-milled yeast (fromComparative Example C1) in a Hobart single-screw mixer in the presenceof grinding agent increases the disruption of the yeast cells to 38%.

Example 2 shows that further mixing of air-milled and Hammer-milledyeast with grinding agent (from Comparative Example C2A) in a Hobartsingle-screw mixer increases the disruption of the yeast cells to 57%.

Comparative Examples C3 and C4 show that in the absence of grindingagent and with or without Hammer-milling (respectively), using twinscrew extrusion with a compression zone, the yeast cell disruption wasgreater than 80%.

TABLE 7 Comparison Of Yeast Cell Disruption Efficiency Free OilDisruption Example % DWT Efficiency, % C1 8 33 C2A* 12.6 62 1* 9.2 38 2*13.8 57 C3 19.6 82 C4 21 87 *The free oil liberated is normalized usingthe actual weight fraction of biomass in the pellet in Example 1,Example 2 and Comparative Example C2A.

SCF Extraction

The extraction vessel was charged with approximately 25 g (yeast basis)of disrupted yeast biomass from Comparative Examples C1, C2A and C4,respectively. The yeast were flushed with CO₂, then heated toapproximately 40° C. and pressurized to approximately 311 bar. The yeastwere extracted at these conditions at a flow rate of 4.3 g/min CO₂ forapproximately 6.7 hr, giving a final solvent-to-feed (S/F) ratio ofabout 75 g CO₂/g yeast. Extraction yields are reported in the Tablebelow.

The data show that higher cell disruption leads to significantly higherextraction yields, measured as the weight percent of crude extractedoil.

TABLE 8 Comparison Of Cell Disruption Efficiency And Oil Extraction Ex-Yeast Cell S/F tracted Charge disruption ratio Oil (g Dry efficiencyTemp. Pressure Time (g CO₂/ Yield Example weight) (%) (° C.) (bar) (hr)g yeast) (wt %) C1 25.1 33 40 310 6.6 74.7 7.5 C2A 25.0 52 40 311 6.876.7 8.9 C4 25.2 87 41 310 6.7 74.4 18.8

Comparative Examples C5A, C5B, C5C, C6A, C6B And C6C Comparison of Meansto Create a Disrupted Biomass Mix from Yarrowia lipolytica

Comparative Examples C5A, C5B, C5C, C6A, C6B and C6C describe a seriesof comparative tests performed to prepare disrupted yeast powder,wherein the initial microbial biomass was either drum dried flakes orspray-dried powder of yeast, mixed with or without a grinding agent in atwin-screw extruder.

In each of Comparative Examples C5A, C5B, C5C, C6A, C6B and C6C, theinitial yeast biomass was from Yarrowia lipolytica strain Y9502, havinga moisture level of 2.8% and containing approximately 36% total oil. Thedried yeast flakes or powder (with or without grinding agent) were fedto an 18 mm twin screw extruder (Coperion Werner Pfleiderer ZSK-18 mmMC) operating with a 10 kW motor and high torque shaft, at 150 rpm. Theresulting disrupted yeast powder was cooled in a final water cooledbarrel.

The disrupted yeast powder prepared in Comparative Examples C5A, C5B,C5C, C6A, C6B and C6C was then subjected to supercritical CO₂ extractionand total extraction yields were compared.

Comparative Example C5A Drum-Dried Yeast Flakes without Grinding Agent

Drum dried flakes of yeast biomass were fed at 2.3 kg/hr to the twinscrew extruder operating with a % torque range of 34-35. The disruptedyeast powder was cooled to 27° C.

Comparative Example C5B Drum-Dried Yeast Flakes with Grinding Agent

92.5 parts of drum dried flakes of yeast biomass were premixed in a bagwith 7.5 parts of Celite 209 D-earth. The resultant dry mix was fed at2.3 kg/hr to the twin screw extruder operating with a % torque range of44-47. The disrupted yeast powder was cooled to 29° C.

Comparative Example C5C Drum-Dried Yeast Flakes with Grinding Agent

85 parts of drum dried flakes of yeast biomass were premixed in a bagwith 15 parts of Celite 209 D-earth. The resultant dry mix was fed at2.3 kg/hr to the twin screw extruder operating with a % torque range of48-51. The disrupted yeast powder was cooled to 29° C.

Comparative Example C6A Spray-Dried Yeast Powder without Grinding Agent

Spray dried powder of yeast biomass were fed at 1.8 kg/hr to the twinscrew extruder operating with a % torque range of 33-34. The disruptedyeast powder was cooled to 26° C.

Comparative Example C6B Spray-Dried Yeast Powder with Grinding Agent

92.5 parts of spray dried powder of yeast biomass were premixed in a bagwith 7.5 parts of Celite 209 D-earth. The resultant dry mix was fed at1.8 kg/hr to the twin screw extruder operating with a % torque range of37-38. The disrupted yeast powder was cooled to 26° C.

Comparative Example C6C Spray-Dried Yeast Powder with Grinding Agent

85 parts of spray dried powder of yeast biomass were premixed in a bagwith 15 parts of D-earth (Celite 209). The resultant dry mix was fed at1.8 kg/hr to the twin screw extruder operating with a % torque range of38-39. The disrupted yeast powder was cooled to 27° C.

SCF Extraction

The extraction vessel was charged with 11.7 g (yeast basis) of disruptedyeast biomass from Comparative Examples C5A, C5B, C5C, C6A, C6B and C6C,respectively. The yeast was flushed with CO₂, then heated toapproximately 40° C. and pressurized to approximately 311 bar. The yeastsamples were extracted at these conditions at a flow rate of 4.3 g/minCO₂ for 3.2 hr, giving a final solvent-to-feed (S/F) ratio ofapproximately 75 g CO₂/g yeast. Extraction yields for variousformulations are reported in Table 9.

The data show that samples having D-earth as a grinding agent (i.e.,Comparative Examples C5B, C5C, C6B and C6C) lead to higher extractionyields than those wherein D-earth was not present (i.e., ComparativeExamples C5A and C6A).

TABLE 9 Comparison Of Oil Extraction Of Disrupted Yeast With And WithoutGrinding Agent Ex- Yeast S/F tracted Charge CO₂ Flow ratio Oil (g DryTemp. Pressure Rate Time (g CO₂/ Yield Example weight) (° C.) (bar)(g/min) (hr) g yeast) (wt %) C5A 11.7 40 311 4.3 3.2 76.4 31.8 C5B 11.741 312 4.3 3.2 76.6 35.4 C5C 11.7 40 312 4.3 3.2 76.7 35.1 C6A 11.7 40311 4.3 3.2 76.4 30.5 C6B 11.7 40 311 4.3 3.2 76.6 37.9 C6C 11.7 40 3114.3 3.2 76.7 38.8

Examples 3, 4, 5, 6, 7, 8, 9 And 10 Comparison of Means to Create SolidPellets from Yarrowia lipolytica

Examples 3-10 describe a series of comparative tests performed to mixspray dried powder or drum-dried flakes of yeast biomass with a grindingagent and binding agent, to provide solid pellets comprising disruptedmicrobial biomass.

In each of Examples 3-10, the initial yeast biomass was from Yarrowialipolytica strain Y9502, having a moisture level of 2.8% and containingapproximately 36% total oil. Following preparation of solid pellets,approximately 1 mm diameter×2 to 8 mm in length, the pellets weresubjected to supercritical CO₂ extraction and total extraction yieldswere compared. Mechanical compression properties and attritionresistance of the solid pellets were also analyzed.

Example 3

85 parts of spray dried powder of yeast biomass were premixed in a bagwith 15 parts of Celatom MN-4 D-earth. The resultant dry mix was fed at2.3 kg/hr to an 18 mm twin screw extruder (Coperion Werner PfleidererZSK-18 mm MC). Along with the dry feed, a water/sugar solution made of14 parts water and 5.1 parts sugar was injected after the disruptionzone of the extruder at a flow-rate of 8.2 ml/min. The extruder wasoperating with a 10 kW motor and high torque shaft, at 150 rpm and %torque range of 58-60 to provide a disrupted yeast powder cooled to 24°C. in a final water cooled barrel.

The fixable mix was then fed into a MG-55 LCI Dome Granulator assembledwith 1 mm hole diameter by 1 mm thick screen and set to 70 RPM.Extrudates were formed at 67.5 kg/hr and a steady 2.7 amp current. Thesample was dried in a Sherwood Dryer for 10 min to provide solid pelletshaving a final moisture level of 7.1%.

Example 4

A fixable mix prepared according to Example 3 was passed through agranulator at 45 RPM. Extrudates were formed at 31.7 kg/hr and dried ina Sherwood Dryer for 10 min to provide solid pellets having a finalmoisture level of 8.15%.

Example 5

A fixable mix prepared according to Example 3 was passed through agranulator at 90 RPM. Extrudate pellets were dried in a MDB-400 FluidBed Dryer for 15 min to provide solid pellets having a final moisturelevel of 4.53%.

Example 6

85 parts of spray dried powder of yeast biomass were premixed in a bagwith 15 parts of Celatom MN-4 D-earth. The resultant dry mix was fed at2.3 kg/hr to an 18 mm twin screw extruder (Coperion Werner PfleidererZSK-18 mm MC) operating with a 10 kW motor and high torque shaft, at 150rpm and % torque range of 70-74 to provide a disrupted yeast powdercooled to 31° C. in a final water cooled barrel.

The disrupted yeast powder was then mixed in a Kitchen Aid mixer with a22.6% solution of sucrose and water (i.e., 17.5 parts water and 5.1parts sugar). The total mix time was 4.5 min with the solution addedover the first 2 min.

The fixable mix was fed to a MG-55 LCI Dome Granulator assembled with 1mm hole diameter by 1 mm thick screen and set to 70 RPM. Extrudates wereformed at 71.4 kg/hr and a steady 2.7 amp current. The sample was driedin a Sherwood Dryer for a total of 20 min to provide solid pelletshaving a final moisture level of 6.5%.

Example 7

Disrupted yeast powder prepared according to Example 6 was placed in aKDHJ-20 Batch Sigma Blade Kneader with a 22.6% solution of sucrose andwater (i.e., 17.5 parts water and 5.1 parts sugar). The total mix timewas 3.5 min with the solution added over the first 2 min.

The fixable mix was fed to a MG-55 LCI Dome Granulator assembled with 1mm hole diameter by 1 mm thick screen and set to 90 RPM. Extrudates wereformed at 47.5 kg/hr and a steady 2.3 amp current. The sample was driedin a Sherwood Dryer for a total of 15 min to provide solid pelletshaving a final moisture level of 7.4%.

Example 8

Drum dried flakes of yeast biomass were fed at 1.8 kg/hr to an 18 mmtwin screw extruder (Coperion Werner Pfleiderer ZSK-18 mm MC) operatingwith a 10 kW motor and high torque shaft, at 150 rpm and % torque rangeof 38-40 to provide a disrupted yeast powder cooled to 30° C. in a finalwater cooled barrel.

The disrupted yeast powder (69.5 parts) was mixed in a Kitchen Aid mixerwith 12.2% Celite 209 D-earth (12.2 parts) and an aqueous sucrosesolution (18.3 parts) made from a 3.3 ratio of water to sugar. The totalmix time was 4.5 min with the solution added over the first 2 min.

The fixable mix was fed to a MG-55 LCI Dome Granulator assembled with 1mm hole diameter by 1 mm thick screen and set to 90 RPM. Extrudates wereformed at 68.2 kg/hr and a steady 2.5 amp current. The sample was driedin a Sherwood Dryer for a total of 15 min to provide solid pelletshaving a final moisture level of 6.83%.

Example 9

Drum dried flakes of yeast biomass (85 parts) were premixed in a bagwith 15 parts of Celite 209 D-earth. The resultant dry mix was fed at2.3 kg/hr to an 18 mm twin screw extruder (Coperion Werner PfleidererZSK-18 mm MC). Along with the dry feed, a water/sugar solution made of14 parts water and 5.1 parts sugar was injected after the disruptionzone of the extruder at a flowrate of 8.2 ml/min. The extruder wasoperating with a 10 kW motor and high torque shaft, at 150 rpm and %torque range of 61-65 to provide a disrupted yeast powder cooled to 25°C. in a final water cooled barrel.

The fixable mix was then fed into a MG-55 LCI Dome Granulator assembledwith 1 mm hole diameter by 1 mm thick screen and set to 90 RPM.Extrudates were formed at 81.4 kg/hr and a steady 2.5 amp current. Thesample was dried in a Sherwood Dryer for 15 min to provide solid pelletshaving a final moisture level of 8.3%.

Example 10

Drum dried flakes of yeast biomass (85 parts) were premixed in a bagwith 15 parts of Celatom NM-4 D-earth. The resultant dry mix was fed at4.6 kg/hr to an 18 mm twin screw extruder (Coperion Werner PfleidererZSK-18 mm MC). Along with the dry feed, a water/sugar solution made of14 parts water and 5.1 parts sugar was injected after the disruptionzone of the extruder at a flowrate of 8.2 ml/min. The extruder wasoperating with a 10 kW motor and high torque shaft, at 300 rpm and %torque of about 34 to provide a disrupted yeast powder.

The fixable mix was then fed into a MG-55 LCI Dome Granulator assembledwith 1 mm hole diameter by 1 mm thick screen and set to 90 RPM.Extrudate was formed at 81.4 kg/hr and a steady 2.5 amp current. Thesample was dried in a Sherwood Dryer for 15 min to provide solidpellets.

Compression Testing and Attrition Resistance of Solid Pellets

Compression testing was performed as follows. The testing apparatus andprotocol described in ASTM standard D-6683 was used to assess theresponse of solid pellets to external loads, such as that imposed by agas pressure gradient. In the test, the volume of a known mass ismeasured as a function of a mechanically applied compaction stress. Asemi-log graph of the results typically is a straight line with a slope,β, reflecting the compression of the sample. Higher values of β reflectgreater compression. This compression can be indicative of particlebreakage, which would lead to undesirable segregation and gas flowrestriction in processing.

At the conclusion of the ASTM test, the load was maintained on thepellets an additional 2 hrs, simulating extended processing time. Creep,measured after 2 hrs, is a further indication of the likelihood of thesolid pellet to deform. Lower creep indicates less deformation.

The test cell containing the sample was then inverted, and the pelletsample was poured out. If necessary, the cell was gently tapped torelease the contents. The ease of emptying the cell and the resultanttexture (i.e., loose or agglomerated) of the pellets was noted.

The texture after the test is a qualitative observation of how hard itwas to empty the test cell used in the previous measurements. The mostdesirable samples poured out immediately, while some required increasingamounts of tapping, and may have fallen out in large chunks (i.e., lessdesirable).

To determine attrition resistance, solid pellets (10 g) previouslycompressed in the Compression Testing ASTM test were then transferred toa 3″ diameter, 500 micron sieve. The sieve was tapped by hand to removeany initial fragments of pellets smaller than 500 microns. The netweight of remaining pellets was noted. Then three cylindrical grindingmedia beads, each 0.50″ diameter by 0.50″ thick, weighing 5.3 gramseach, were added to the sieve. The sieve was placed in an automaticsieve shaker (Gilson Model SS-3, with a setting of “8”, with automatictapping “on”) and shaken for periods of 2, 5 or 10 min. The grindingmedia beads repeatedly strike the pellets from random angles. Aftershaking, the pan under the sieve was weighed to determine the amount ofmaterial that had been attrited and had fallen through the sieve. Thistest is intended to simulate very rough handling of the pellets afterthe oil extraction process.

Solid pellets from Examples 3-10, respectively, were analyzed todetermine their compression properties and attrition resistance. Resultsare tabulated below in Table 10.

TABLE 10 Mechanical Compression And Attrition Of Solid Pellets LooseCreep Attrition Bulk Com- after 2 hr In sieving Density pression at 1994Texture 2 min 10 min Example lb/ft³ Exponent β lb/ft² (%) after test (%)(%) 10 28.98 0.06857 12.78 Puck, 2.9 11.8 breaks into 5 pieces 3 31.270.05335 6.95 No puck 20.5 99.0 4 31.85 0.05966 13.07 Many taps, 8.8 47.45 pieces 5 24.66 0.03928 4.10 Two taps, 8.8 42.1 loose 6 30.63 0.047468.34 Few taps, 10.0 48.7 loose 7 28.89 0.04347 3.11 Few taps, 9.1 43.1loose 8 28.35 0.02976 0.00 Loose 5.2 22.4 9 31.66 0.07730 16.06 Puck 7.536.2

SCF Extraction

The extraction vessel was charged with solid pellets (on a dry weightbasis, as listed in Table 11) from Examples 3-9, respectively. Thepellets were flushed with CO₂, then heated to about 40° C. andpressurized to approximately 311 bar. The pellets were extracted atthese conditions at a flow rate of 4.3 g/min CO₂ for about 6.8 hr,giving a final solvent-to-feed (S/F) ratio of approximately 150 g CO₂/gyeast. In some Examples a second run was performed for an additional 4.8hrs, such that the total time for extraction was 11.6 hr. The oilextraction yields and specific parameters used for extraction are listedin Table 11.

TABLE 11 Comparison Of Oil Extraction Of Solid Pellets Yeast CO₂ S/F Ex-Charge Flow ratio tracted (g Dry Temp. Pressure Rate Time (g CO₂/ OilYield Example weight) (° C.) (bar) (g/min) (hr) g yeast) (wt %) 3 12.8  40 311 4.3 6.8 150 37.3   4 21.5 ^(b) 40 312 4.3 11.6 151 39.3 ^(a) 512.9   40 312 4.3 6.9 150 36.4   6 12.8   41 311 4.3 6.8 149 36.6   721.7 ^(b) 40 312 4.3 11.6 150 37.4 ^(a) 8 21.8 ^(b) 40 311 4.3 11.6 15031.0 ^(a) 9 12.6   41 312 4.3 6.8 152 39.1   ^(a) average result fromtwo runs ^(b) sum of two runs

Compression Testing and Attrition Resistance of Residual Pellets(Post-Extraction)

Following SCF extraction, the residual pellets from Examples 3-9,respectively, were analyzed to determine their compression propertiesand attrition resistance. Results are tabulated below in Table 12.

TABLE 12 Mechanical Compression And Attrition Of Residual Pellets(Post-Extraction) Creep Loose after 2 hr Attrition Bulk at 1994 Insieving Density Compression lb/ft² Texture 2 min 5 min Example lb/ft³Exponent β (%) after test (%) (%) 3 23.64 0.03352 0.75 Loose n/a  73.0*4 23.50 0.02035 0.78 One tap, 12.0 28.6 loose 5 24.14 0.02636 0.71 Loose10.9 26.4 6 24.66 0.02002 0.62 Loose 10.6 27.1 7 21.78 0.02897 0.98 Onetap, 10.9 25.5 loose 8 21.84 0.02821 0.67 Loose 7.7 18.5 9 23.87 0.022460.58 Loose 10.1 22.3 *The expected attrition from 5 minutes of sievingwas estimated by interpolating the results of a 2 minute test and a 6.5minute test

Based on the above, it is concluded that the process described herein[i.e., comprising steps of (a) mixing a microbial biomass, having amoisture level and comprising oil-containing microbes, and at least onegrinding agent capable of absorbing oil, to provide a disrupted biomassmix; (b) blending at least one binding agent with said disrupted biomassmix to provide a fixable mix capable of forming a solid pellet; and (c)forming said solid pellet from the fixable mix] can be successfullyutilized to produce solid pellets comprising disrupted microbial biomassfrom Yarrowia lipolytica. Furthermore, the present Example demonstratesthat the solid Yarrowia lipolytica pellets can be extracted with asolvent (i.e., SCF extraction) to provide an extract comprising themicrobial oil.

Example 11 A. Method for Determining Lipid Distribution for Yeast CellBiomass, Oil, and Residual Biomass Samples

Yeast cell samples and residual biomass samples (i.e., yeast cells afterextraction with CO₂) were extracted using a modification of the methodof Bligh & Dyer (based on procedures outlined in Lipid Analysis, W. W.Christie 2003), separated with thin-layer chromatography (TLC) anddirectly esterified/transesterified using methanolic hydrogen chloride.Oil samples were dissolved in chloroform/methanol, then separated withTLC and directly esterified/transesterified. Theesterified/transesterified samples were analyzed by gas chromatography.

Yeast cell and residual biomass samples were received as a dry powder. Apredetermined portion (100-200 mg or less, depending on the PUFAconcentration) of the sample was weighed into a 13×100 mm glass testtube with a Teflon™ cap to which 3 mL volume of a 2:1 (volume:volume)methanol/chloroform solution was added. The sample was vortexedthoroughly and incubated at room temperature for one hr with gentleagitation and inversion. After the hr, 1 mL of chloroform and 1.8 mL ofdeionized water were added, the mixture was agitated and thencentrifuged to separate the two layers that formed. Using a pasteurpipette, the bottom layer was removed into a second, tared 13 mm glassvial and the aqueous top layer was re-extracted with a second 1 mLportion of chloroform for 30 min. The two extracts were combined andconsidered as the “first extract”. The solvent was removed using aTurboVap™ at 50° C. with dry nitrogen and the remaining oil wasresuspended in the appropriate amount of 6:1 (volume:volume)chloroform/methanol to obtain a 100 mg/mL solution.

The oil obtained as described above (for yeast cell and residual biomasssamples) and the oil samples from CO₂ extraction of the yeast cells wereanalyzed by TLC. The TLC was typically done using one tank, although atwo tank procedure was also employed when individual PLs were to beidentified. In the one tank TLC procedure, a 5×20 cm silica gel 60 plate(EMD #5724-3, obtained from VWR) was prepared by drawing a light pencilline all the way across the plate 2 cm from the bottom. An appropriateamount of sample, (˜60 μL) was spotted completely across the plate ontop of the pencil line without leaving any space between the spots. Asecond plate was spotted with known standards and the sample using 1-2μL amounts. The plates were air dried for 5-10 min and developed using ahexane-diethyl ether-acetic acid mixture (70:30:1 by volume) that hadbeen equilibrated in the tank for at least 30 min with a piece ofblotting paper prior to running the plate.

After the plates had been developed to within a ¼inch of the top, theywere dried in a N₂ environment for 15 min. The second plate, with thestandards and small sample spot, was then developed in a tank that hadbeen saturated with iodine crystals to serve as a reference for thepreparative plate. The bands on the preparative plate were identified byvery lightly staining the edge of the bands with iodine and, using apencil, grouping the bands according to each fraction (i.e., the PLfraction, FFA fraction, TAG fraction and DAG fraction). The DAG band canshow some separation between the 1,2-DAGs, the 1,3-DAGs, and the MAGband, and typically this entire area was cut out as the DAG band. Thebands were cut out of the gel and transferred to a 13 mm glass vial. Theremainder of the plate was developed in the iodine tank to verifycomplete removal of the bands of interest.

To the glass vial containing each band, an appropriate amount oftriglyceride internal standard in toluene was added. Depending on thevisible concentration of each band, 100 μL of a 0.1 to 5 mg/mL internalstandard was usually used. The co-solvent (in this case, toluene) wasadded with the internal standard. If an internal standard was not used,additional co-solvent was added to complete theesterification/transesterification of the longer chain lipids. 1 mL of a1% methanolic hydrogen chloride solution (prepared by slowly adding 5 mLacetyl chloride to 50 mL cooled, dry methanol) was added, the samplecapped, gently mixed, and placed in a heating block at 80° C. for onehr. After one hr, the sample was removed and allowed to cool. 1 mL of a1 N sodium chloride solution and 400 μL of hexane were added, the samplethen vortexed for at least 12 seconds and centrifuged to separate thetwo layers. The top layer was then removed, with care being taken to notcontaminate it with any of the aqueous (bottom) layer. The top layer wasplaced into a GC vial fitted with an insert and capped.

The sample was analyzed using an Agilent Model 6890 Gas Chromatograph(Agilent Technologies, Santa Clara, Calif.), equipped with a FlameIonization Detector (FID) and an Omegawax 320 column (30 m×0.32 mm ID×25μm film thickness and manufactured by Supelco (Bellfonte, Pa.)). Thehelium carrier gas was kept constant within a range of 1-3 mL/min with asplit ratio of 20:1 or 30:1. The oven conditions were as follows:initial temperature of 160° C. with an initial time of 0 min and anequilibration time of 0.5 min. The temperature ramps were 5 degrees/minto 200° C. for a final hold time of 0 min then 10 degrees/min to 240° C.for 4 min of hold time for a total of 16 min. The inlet was set to 260°C. The FID detector was also set to 260° C. A Nu-Chek Prep GLC referencestandard (#461) was run for retention time verification.

The GC results were collected using Agilent's Custom Reports and thearea of each fatty acid was transferred to an Excel spreadsheet forcalculation of their percentages. Correction factors to convert thetotal amount of fatty acids in a lipid class could then be applied.Total percentages of each component were compared to the derivatizedoriginal extract prior to TLC.

B. Extraction Method

Extraction was performed according to the GENERAL METHODS. Analyses ofthe various lipid components in the yeast and extracted oils reported inExamples 12-20 below were determined using the thin layer and gaschromatographic methods described herein above. For yeast samples, thissummary reflects analysis of the lipids extracted from the sample usingthe analytical procedure. The amount of lipids analyzed by thisprocedure for the Extracted Yeast samples is relatively small whencompared to that of the comparable Feed Yeast and Oil Extract samples(typically <3% of the extractable oils in the starting feed yeast). Thesummary tables of results show the relative distribution of lipidcomponents for each of the samples. For each identified lipid componentshown in the horizontal row across the top of the table, the relativedistribution of that component as phospholipids (PL), diacylglycerides(DAG), free fatty acids (FFA), and triacylglycerides (TAG) is shownvertically down the table columns. The first line for each sample showsanalysis of the derivatized original extract prior to TLC. The followinglines give the analyses of each component by TLC and GC, with the totalpercentages of each component presented in the last line for thatsample.

In the Examples 12-20, infra, the reported extraction yield of oil wasdetermined by the weight difference between the yeast sample beforeextraction and the residual biomass after extraction, expressed as apercentage. The weight difference was assumed to be due to the amount ofoil extracted by contacting with CO₂. The actual weight of the oilobtained was generally found to be within about 85% of the weightexpected based on the mass difference.

Example 12 Extraction Curve at 311 Bar and 40° C.

The purpose of this Example was to demonstrate generation of anextraction curve. An 8-mL extraction vessel fabricated from 316 SStubing (0.95 cm o.d.×0.62 cm i.d.×26.7 cm long) was repeatedly chargedwith nominally 2.7 g of dried and mechanically disrupted yeast cells(i.e., Yarrowia lipolytica strain Y8672) for a series of extractions todetermine the extraction curve for this yeast sample at 40° C. and 311bar. For each extraction, the extraction vessel and yeast were flushedwith CO₂ and then pressurized to 311 bar with CO₂ at 40° C. The yeastsample was extracted at these conditions and a CO₂ flow rate of 1.5g/min for various times to give a range of solvent-to-feed ratiosresulting in a corresponding extraction yield, as shown in Table 13.FIG. 4 plots these data in an extraction curve. The break in the curveat a solvent-to-feed ratio of about 40 g CO₂/g yeast indicates that atleast this solvent ratio is required to effectively extract theavailable oil in this particular yeast sample at the selectedtemperature and pressure.

The series of extractions can be repeated at different temperatureand/or pressure conditions to generate a series of extraction curves fora particular microbial biomass sample, enabling selection of the optimumextraction conditions based on economics, desired extraction yield, ortotal amount of CO₂ used, for example.

TABLE 13 Solvent To Feed Ratio And Extraction Yield Data At 311 Bar And40° C. Specific Solvent Extraction Ratio Yield (g CO₂/g Yeast) (wt %)6.0 5.5 6.0 6.2 6.0 4.7 10.9 10.3 13.6 9.3 14.8 10.9 19.5 13.0 19.7 10.619.7 15.5 24.8 14.3 25.1 17.5 25.7 16.8 29.9 18.0 39.5 18.7 49.5 18.754.5 18.8 59.8 18.9 80.5 19.0 98.5 18.7 109.3 18.7 149.8 19.2

Comparative Example C7 Extraction of Yeast Cells without Fractionationof the Extract Obtained

The purpose of this Comparative Example was to demonstrate extraction ofmicrobial biomass with CO₂, without fractionation of the extract orsequential extraction of the residual biomass, and the lipid compositionof the extract so obtained. An 18-mL extraction vessel fabricated from316 SS tubing (1.27 cm o.d.×0.94 cm i.d.×26.0 cm long) was charged with4.99 g of dried and mechanically disrupted yeast cells (i.e., Yarrowialipolytica strain Y8672). The yeast sample was flushed with CO₂, thenheated to 40° C. and pressurized to 222 bar. The yeast sample wasextracted at these conditions at a flow rate of 2.3 g/min CO₂ for 5.5hrs, giving a final solvent-to-feed ratio of 149 g CO₂/g yeast. Theyield of the extract was 18.2 wt %.

The Table below summarizes lipid analyses for the starting feed yeast(the microbial biomass), the extracted yeast (the residual biomass), andthe extract obtained. For the yeast cells, 50 weight percent (wt %) ofthe FFAs and 59.8 wt % of the TAGs were found to contain EPA, calculatedrespectively as the percentage of 3/6 [the wt % of FFAs comprising EPAin the feed yeast divided by the total wt % of FFAs in the feed yeast,expressed as a percentage and with both percent values taken from theTLC analysis] and as the percentage of 49/82 [the wt % of TAGscomprising EPA in the feed yeast divided by the total wt % of TAGs inthe feed yeast, expressed as a percentage and with both percent valuestaken from the TLC analysis]. The absence (0 wt %) of PLs in the extractshow that the PL fraction of the lipids present in the starting feedyeast remains in the residual biomass and does not partition with theCO₂ into the extract. The results also show the extract contains 90 wt %TAGs, 4 wt % FFAs, and 6 wt % DAGs. For the extract, 50% of the FFAs and58.9% of the TAGs were found to contain EPA, calculated respectively asthe percentage of 2/4 [the wt % of FFAs comprising EPA in the extractdivided by the total wt % of FFAs in the extract, expressed as apercentage and with both percent values taken from the TLC analysis] andthe percentage of 53/90 (the wt % of TAGs comprising EPA in the extractdivided by the total wt % of TAGs in the extract, expressed as apercentage, with both percent values taken from the TLC analysis).

TABLE 14 Comparative Example C7: Weight Percent Distribution of LipidComponents 18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4(n-3) 20:5 Sample Palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETAEPA other Total Feed Yeast 3 2 4 15 1 2 2 1 2 55 11 100 PL 1 0 0 1 0 0 00 0 2 1 6 DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 3 1 6 TAG 12 4 13 1 1 1 1 1 49 7 82 3 2 5 16 1 2 2 1 1 56 9 100 Residual Biomass 74 4 18 1 2 2 1 1 49 10 100 PL 6 3 2 10 1 1 1 0 1 14 5 43 DAG 0 0 0 1 0 00 0 0 2 1 4 FFA 1 0 0 1 0 0 0 0 0 6 1 12 TAG 1 1 2 7 1 1 1 1 0 23 4 41 84 4 18 1 2 2 1 2 45 11 100 Extract 2 2 4 14 1 2 3 3 1 56 10 100 PL 0 0 00 0 0 0 0 0 0 0 0 DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 2 14 TAG 1 2 4 14 1 2 2 1 1 53 8 90 2 2 5 16 1 2 2 1 1 58 9 100

Example 13 Lipid Fractionation by Sequential Pressure Extraction

The purpose of this Example was to demonstrate sequential pressureextraction of a yeast sample and the lipid compositions of the extractsobtained. An 18-mL extraction vessel fabricated from 316 SS tubing (1.27cm o.d.×0.94 cm i.d.×26.0 cm long) was charged with 3.50 g of dried andmechanically disrupted yeast cells (i.e., Yarrowia lipolytica strainY8672).

Extract A: The yeast was flushed with CO₂, then heated to 40° C. andpressurized to 125 bar. The yeast sample was extracted at theseconditions at a flow rate of 2.3 g/min CO₂ for 5 hr, at which time thepressure was increased to 150 bar. The extraction was continued for anadditional 1.2 hr, giving a final solvent-to-feed ratio of 238 g CO₂/gyeast. The yield of Extract A was 11.7 wt %.

Extract B: The extraction was continued with the same partiallyextracted yeast sample by increasing the pressure to 222 bar andcontinuing the CO₂ flow at 2.3 g/min for 4.0 hr, giving a finalsolvent-to-feed ratio of 153 g CO₂/g yeast for this fraction. The yieldof Extract B was 6.2 wt % of the original yeast charged to theextraction vessel.

The Table below summarizes lipid analyses for the starting feed yeastand the two extracts. The results show that under the extractionconditions employed, the FFAs and DAGs of the microbial biomass oilselectively partitioned into Extract A (which contains 9 wt % of each),while Extract B was enriched in TAGs (and contains only 1 wt % DAGs andno measured FFAs). More specifically, Extract B was about 99% TAGs, andabout 62.6% of the TAGs were found to contain EPA (calculated as thepercentage of 62/99 [the wt % of TAGs comprising EPA in Extract Bdivided by the total wt % of TAGs in Extract B, expressed as apercentage, and with both percent values taken from the TLC analysis].In contrast, about 59.8% of the TAGs of the yeast cells were found tocontain EPA, calculated as the percentage of 49/82 (the wt % of TAGscomprising EPA in the feed yeast divided by the total wt % of TAGs inthe feed yeast, expressed as a percentage, with both percent valuestaken from the TLC analysis). These results are expected to be similarto the results which could be obtained by SCF CO₂-extraction of theyeast sample to provide an extract which is subsequently fractionatedvia stepwise pressure reduction.

TABLE 15 Example 13: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Total Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPAother % Feed Yeast 3 2 4 15 1 2 2 1 2 55 11 100 PL 1 0 0 1 0 0 0 0 0 2 16 DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 3 1 6 TAG 1 2 4 13 11 1 1 1 49 7 82 Sum 3 2 5 16 1 2 2 1 1 56 9 100 Extract A 3 2 5 16 1 2 21 1 56 10 PL 0 0 0 0 0 0 0 0 0 0 0 0 DAG 1 0 0 2 0 0 0 0 0 3 1 9 FFA 1 00 1 0 0 0 0 0 4 1 9 TAG 2 2 5 15 1 1 1 1 1 44 7 82 Sum 4 3 6 18 1 2 2 11 51 10 100 Extract B 1 2 4 13 1 2 2 1 2 62 9 PL 0 0 0 0 0 0 0 0 0 0 0 0DAG 0 0 0 0 0 0 0 0 0 0 0 1 FFA 0 0 0 0 0 0 0 0 0 0 0 0 TAG 1 2 4 14 1 22 1 1 62 8 99 Sum 1 2 5 14 1 2 2 1 1 62 8 100

Example 14 Lipid Fractionation by Sequential Pressure Extraction

The purpose of this Example was to demonstrate sequential pressureextraction of a yeast sample under different extraction conditions andthe lipid compositions of the extracts obtained. An 89-mL extractionvessel fabricated from 316 SS tubing (2.54 cm o.d.×1.93 cm i.d.×30.5 cmlong) was charged with 15.0 g of dried and mechanically disrupted yeastcells (i.e., Yarrowia lipolytica strain Y8672).

Extract A: The yeast was flushed with CO₂, then heated to 40° C. andpressurized to 125 bar. The yeast sample was extracted at theseconditions at a flow rate of 2.3 g/min CO₂ for 3.9 hr, at which time theflow rate was increased to 4.7 g/min CO₂ and the extraction wascontinued for an additional 2.3 h. The pressure was then increased to141 bar. The extraction was continued for an additional 4.1 hr at 4.7g/min CO₂, giving a final solvent-to-feed ratio of 154 g CO₂/g yeast.The yield of Extract A was 8.7 wt %.

Extract B: The extraction was continued with the same partiallyextracted yeast sample by increasing the pressure to 222 bar andcontinuing the CO₂ flow at 4.7 g/min for 8.0 hr, giving a finalsolvent-to-feed ratio of 150 g CO₂/g yeast for this extract. The yieldof Extract B was 15.4 wt % of the original yeast charged to theextraction vessel.

The Table below summarizes lipid analyses for the starting feed yeast,the residual biomass after both extractions, and the two extracts. Theresults show that the PL fraction of the lipids present in the startingfeed yeast remains in the residual biomass. Under the extractionconditions employed, the FFAs and DAGs of the microbial biomassselectively partitioned into Extract A, while Extract B was enriched inTAGs. More specifically, Extract B was about 97% TAGs with no measuredFFAs, and about 61.9% of the TAGs were found to contain EPA. Incontrast, about 59.8% of the TAGs of the yeast cells were found tocontain EPA. These results are expected to be similar to the resultswhich could be obtained by SCF CO₂-extraction of the yeast sample toprovide an extract which is subsequently fractionated via stepwisepressure reduction.

TABLE 16 Example 14: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal Feed Yeast 3 2 4 15 1 2 2 1 2 55 11 100 PL 1 0 0 1 0 0 0 0 0 2 1 6DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 3 1 6 TAG 1 2 4 13 1 11 1 1 49 7 82 Sum 3 2 5 16 1 2 2 1 1 56 9 100 Residual Biomass 6 3 4 151 3 2 1 3 43 15 100 PL 5 2 1 9 0 0 1 0 1 12 5 37 DAG 0 0 0 1 0 0 0 0 0 21 5 FFA 1 1 0 1 0 1 0 0 0 8 2 15 TAG 1 1 2 7 1 1 1 0 1 24 4 42 Sum 7 4 417 1 2 2 1 2 46 11 100 Extract A 3 2 5 16 1 2 2 1 2 56 9 100 PL 0 0 0 00 0 0 0 0 0 0 0 DAG 1 1 0 2 0 0 0 0 0 4 1 11 FFA 1 0 0 1 0 0 0 0 0 5 1 9TAG 2 2 4 14 1 1 1 1 1 45 7 80 Sum 3 3 5 17 1 2 2 1 1 55 9 100 Extract B1 2 5 14 1 2 2 1 1 61 9 100 PL 0 0 0 0 0 0 0 0 0 0 0 1 DAG 0 0 0 0 0 0 00 0 1 0 2 FFA 0 0 0 0 0 0 0 0 0 0 0 0 TAG 1 2 5 15 1 2 2 1 1 60 8 97 Sum1 2 5 15 1 2 2 1 1 61 8 100

Example 15 Lipid Fractionation by Sequential Pressure Extraction

The purpose of this Example was to demonstrate sequential pressureextraction of a yeast sample under different extraction conditions andthe lipid compositions of the extracts obtained. An 89-mL extractionvessel fabricated from 316 SS tubing (2.54 cm o.d.×1.93 cm i.d.×30.5 cmlong) was charged with 20.0 g of dried and mechanically disrupted yeastcells (i.e., Yarrowia lipolytica strain Y8672).

Extract A: The yeast was flushed with CO₂, then heated to 40° C. andpressurized to 110 bar. The yeast sample was extracted at theseconditions at a flow rate of 4.7 g/min CO₂ for 7.1 hr, giving a finalsolvent-to-feed ratio of 100 g CO₂/g yeast. The yield of Extract A was4.1 wt %.

Extract B: The extraction was continued with the same partiallyextracted yeast sample by increasing the pressure to 222 bar andcontinuing the CO₂ flow at 4.7 g/min for 15.0 hr, giving a finalsolvent-to-feed ratio of 212 g CO₂/g yeast for this extract. The yieldof Extract B was 14.6 wt % of the original yeast charged to theextraction vessel.

The Table below summarizes lipid analyses for the starting feed yeast,the residual biomass after both extractions, and the two extracts. Theresults show that the PL fraction of the lipids present in the startingfeed yeast remains in the residual biomass. Under the extractionconditions employed, the FFAs and DAGs of the microbial biomassselectively partitioned into Extract A, while Extract B was enriched inTAGs (i.e., oil Fraction B was about 95% TAGs). These results areexpected to be similar to the results which could be obtained by SCFCO₂-extraction of the yeast sample to provide an extract which issubsequently fractionated via stepwise pressure reduction.

Examples 13 though 15 herein collectively illustrate that partitioningof the lipid components of the extract can be influenced by theselection of the extraction conditions in a multi-step extraction. Suchpartitioning would likewise result from a sequential reduction ofpressure of the oil extract obtained by a process as illustrated in FIG.3.

TABLE 17 Example 15: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal Feed Yeast 3 2 4 15 1 2 2 1 2 55 11 100 PL 1 0 0 1 0 0 0 0 0 2 1 6DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 3 1 6 TAG 1 2 4 13 1 11 1 1 49 7 82 sum 3 2 5 16 1 2 2 1 1 56 9 100 Residual Biomass 6 3 4 151 2 3 5 2 43 13 100 PL 5 2 2 9 1 1 1 0 1 13 5 41 DAG 0 0 0 1 0 0 0 0 0 21 4 FFA 1 0 0 1 0 0 0 0 0 6 1 12 TAG 1 1 2 7 1 1 1 1 1 25 4 43 sum 7 4 418 1 2 2 1 2 46 11 100 Extract A 5 3 4 15 1 2 2 1 1 56 8 100 PL 0 0 0 00 0 0 0 0 0 0 0 DAG 2 1 1 3 0 0 0 0 0 5 2 13 FFA 2 1 1 3 0 1 1 1 1 22 337 TAG 1 1 3 9 1 1 1 0 1 27 4 49 sum 6 3 5 15 1 2 2 1 1 53 9 100 ExtractB 2 2 5 15 1 2 2 1 1 60 9 100 PL 0 0 0 0 0 0 0 0 0 0 0 0 DAG 0 0 0 1 0 00 0 0 2 0 4 FFA 0 0 0 0 0 0 0 0 0 0 0 1 TAG 1 2 5 16 1 2 2 1 1 56 8 95sum 2 2 5 16 1 2 2 1 1 58 8 100

Example 16 SCF Extraction at 500 Bar

The purpose of this Example was to demonstrate extraction of a yeastsample with CO₂ as a supercritical fluid at 500 bar, and the compositionof the extract obtained. Such extraction conditions could be used in thefirst step of a method for obtaining a refined composition comprising atleast one PUFA, where the method comprises contacting microbial biomasscomprising at least one PUFA with CO₂ under suitable extractionconditions, and subsequently fractionating the extract, for example bysequential pressure reduction.

A 10-mL extraction vessel was charged with 2.01 g of dried andmechanically disrupted yeast cells (i.e., Yarrowia lipolytica strainY4305-F1B1), and the vessel was mounted in an a commercially-availableautomated supercritical fluid extraction instrument, i.e., an Isco ModelSFX3560 extractor. This instrument utilized 10-mL plastic extractionvessels equipped with a 2-micron sintered metal filter on each end ofthe extraction vessel. This vessel was charged with the substrate to beextracted and then loaded into a high pressure extraction chamber whichequalized the pressure on the inside and outside of the extractionvessel. The CO₂ solvent was metered with a syringe pump (ISCO Model260D), preheated to the specified extraction temperature, and thenpassed through the extraction vessel. The extraction chamber was heatedwith electrical resistance heaters to the desired extractiontemperature. Pressure was maintained on the vessel with an automatedvariable restrictor, which was an integral part of the instrument.

The yeast sample was flushed with CO₂, then heated to 40° C. andpressurized to 500 bar. The yeast sample was extracted at theseconditions at a flow rate of 0.86 g/min CO₂ for 5.8 hr, giving a finalsolvent-to-feed ratio of 150 g CO₂/g yeast. The yield of extracted oilwas 32.8 wt %. The Table below summarizes lipid analyses for thestarting feed yeast, the residual biomass after the extraction, and theoil obtained by the extraction. The results show that the PL fraction ofthe lipids present in the starting feed yeast remained in the residualbiomass and did not partition into the CO₂-extracted oil, whichcomprised FFAs, DAGs, and TAGs.

TABLE 18 Example 16: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal Feed Yeast 3 3 6 21 4 4 2 1 2 44 9 100 PL 1 0 0 1 0 0 0 0 0 1 0 4DAG 0 0 0 2 0 0 0 0 0 2 1 6 FFA 0 0 0 1 0 0 0 0 0 2 1 6 TAG 2 2 5 18 3 32 0 1 38 7 84 Sum 3 3 6 22 4 3 2 1 2 43 9 100 Residual Biomass 9 4 5 243 3 2 1 2 36 7 100 PL 7 3 2 14 1 1 1 0 1 11 3 48 DAG 0 0 0 1 0 0 0 0 0 10 4 FFA 1 1 0 2 0 1 0 0 0 4 2 12 TAG 1 1 2 8 1 1 1 0 1 15 3 36 Sum 9 5 525 3 3 2 1 2 32 9 100 Oil 2 2 6 21 4 4 2 1 2 46 9 100 PL 0 0 0 0 0 0 0 00 0 0 0 DAG 0 0 0 2 0 0 0 0 0 2 1 7 FFA 0 0 0 1 0 0 0 0 0 2 1 5 TAG 2 25 19 3 3 2 0 1 40 7 88 Sum 3 3 6 21 4 4 2 1 2 45 9 100

Example 17 SCF Extraction at 310 Bar

The purpose of this Example was to demonstrate extraction of a yeastsample with CO₂ as a supercritical fluid at 310 bar, and the compositionof the extract obtained. Such extraction conditions could be used in thefirst step of a method for obtaining a refined composition comprising atleast one PUFA, where the method comprises contacting microbial biomasscomprising at least one PUFA with CO₂ under suitable extractionconditions, and subsequently fractionating the extract, for example bysequential pressure reduction.

An 89-mL extraction vessel fabricated from 316 SS tubing (2.54 cmo.d.×1.93 cm i.d.×30.5 cm long) was charged with 25.1 g of dried andmechanically disrupted yeast cells (i.e., Yarrowia lipolytica strainY9502). The yeast sample was flushed with CO₂, then heated to 40° C. andpressurized to 310 bar. The yeast sample was extracted at theseconditions at a flow rate of 5.0 mL/min CO₂ for 4.4 hr, giving a finalsolvent-to-feed ratio of 50 g CO₂/g yeast. The yield of extracted oilwas 28.8 wt %. The Table below summarizes lipid analyses for thestarting feed yeast, the residual biomass after the extraction, and theoil obtained by the extraction. The results show that the PL fraction ofthe lipids present in the starting feed yeast remained in the residualbiomass and did not partition into the CO₂-extracted oil, whichcomprised FFAs, DAGs, and TAGs.

TABLE 19 Example 17: Weight Percent Distribution of Lipid Components18:3 20:3 16:0 16:1 18:1 18:2 (n-3) 20:2 (n-6) 20:4 20:5 Sample palmiticPalmitoleic Oleic Linoleic ALA EDA HGLA ARA EtrA EPA other Total FeedYeast 2 1 4 10 1 5 6 2 0 51 12 100 PL 0 0 0 1 0 0 0 0 0 2 1 7 DAG 0 0 01 0 0 0 0 0 3 1 7 FFA 1 0 0 1 0 2 1 0 0 8 1 15 TAG 1 0 3 9 0 2 4 1 0 416 72 Sum 2 1 4 11 1 5 6 1 1 53 9 100 Residual Biomass 4 1 4 12 0 5 6 2 348 10 100 PL 3 0 2 6 0 2 2 0 1 14 5 40 DAG 0 0 1 1 0 1 1 0 0 3 1 9 FFA 00 0 0 0 2 1 0 0 6 2 14 TAG 0 0 2 4 0 2 3 1 0 20 4 38 Sum 4 1 5 12 1 6 62 2 44 12 100 Oil 2 1 4 11 1 5 6 1 0 56 9 100 PL 0 0 0 0 0 0 0 0 0 0 0 1DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 1 0 0 1 0 2 1 0 0 8 2 16 TAG 1 1 4 10 12 4 1 0 43 7 77 Sum 2 1 4 12 1 5 6 1 0 53 9 100

Example 18 SCF Extraction at 222 Bar

The purpose of this Example was to demonstrate extraction of a yeastsample with CO₂ as a supercritical fluid at 222 bar, and the compositionof the extract obtained. Such extraction conditions could be used in thefirst step of a method for obtaining a refined composition comprising atleast one PUFA, where the method comprises contacting microbial biomasscomprising at least one PUFA with CO₂ under suitable extractionconditions, and subsequently fractionating the extract, for example bysequential pressure reduction.

An 89-mL extraction vessel fabricated from 316 SS tubing (2.54 cmo.d.×1.93 cm i.d.×30.5 cm long) was charged with 25.1 g of dried andmechanically disrupted yeast cells (i.e., Yarrowia lipolytica strainY8672). The yeast sample was flushed with CO₂, then heated to 40° C. andpressurized to 222 bar. The yeast sample was extracted at theseconditions at a flow rate of 4.7 g/min CO₂ for 13.7 hr, giving a finalsolvent-to-feed ratio of 154 g CO₂/g yeast. The yield of extracted oilwas 18.1 wt %. This extraction was replicated an additional four times,each time with a fresh yeast sample, and the five extracts consolidated.The residual biomass samples and the extracted oil samples were alsoeach consolidated and mixed to provide composite samples from the fiveextractions. The Table below summarizes lipid analyses for the startingfeed yeast, the consolidated residual biomass, and the consolidated oilobtained by the extraction. The oil was found to comprise 7 wt % FFAs, 7wt % DAGs, and 86 wt % TAGs.

TABLE 20 Example 18: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal Feed Yeast 3 3 4 15 1 2 4 1 3 51 11 100 PL 1 0 0 2 0 0 0 0 0 2 1 8DAG 1 0 0 1 0 0 0 0 0 2 1 6 FFA 0 0 0 0 0 0 0 0 0 3 1 7 TAG 1 2 4 12 1 23 1 2 45 7 79 Sum 3 3 5 16 1 2 3 1 3 53 9 100 Residual Biomass 9 4 4 181 2 3 1 4 36 14 100 PL 8 4 3 14 1 1 2 0 2 16 8 59 DAG 0 0 0 1 0 0 0 0 01 1 3 FFA 1 0 0 1 0 0 0 0 0 5 2 11 TAG 1 1 1 4 0 1 1 0 1 13 4 27 Sum 105 5 19 1 2 3 1 3 36 14 100 Oil 2 2 4 14 1 2 4 1 4 52 12 100 PL 0 0 0 0 00 0 0 0 0 0 0 DAG 1 0 0 1 0 0 0 0 0 3 1 7 FFA 0 0 0 0 0 0 0 0 0 3 1 7TAG 2 2 4 13 1 2 3 1 2 48 7 86 Sum 3 3 5 15 1 3 3 1 3 54 9 100

Example 19 Liquid CO₂ Extraction at 85 Bar

The purpose of this Example was to demonstrate extraction of a yeastsample with CO₂ as a liquid at 85 bar, and the composition of theextract obtained. Such extraction conditions could be used in the firststep of a method for obtaining a refined composition comprising at leastone PUFA, where the method comprises contacting microbial biomasscomprising at least one PUFA with CO₂ under suitable extractionconditions, and subsequently fractionating the extract, for example bysequential pressure reduction.

An 8-mL extraction vessel fabricated from 316 SS tubing (0.95 cmo.d.×0.62 cm i.d.×26.7 cm long) was charged with 0.966 g of dried andmechanically disrupted yeast cells (i.e., Yarrowia lipolytica strainY8672). The yeast sample was flushed with CO₂, and then pressurized to85 bar with liquid CO₂ at 22° C. The yeast sample was extracted at theseconditions at a flow rate of 0.69 g/min CO₂ for 8.5 hr, giving a finalsolvent-to-feed ratio of 361 g CO₂/g yeast. The yield of extracted oilwas 21.4 wt %. The Table below summarizes lipid analyses for thestarting feed yeast, the residual biomass, and the oil obtained by theextraction. The results show that the PL fraction of the lipids presentin the starting feed yeast remained in the residual biomass and did notpartition into the CO₂-extracted oil. The oil comprised 8 wt % FFAs, 5wt % DAGs, and 86 wt % TAGs.

TABLE 21 Example 19: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal Feed Yeast 2 2 4 13 1 3 4 1 2 55 11 100 PL 1 0 0 1 0 0 0 0 0 2 1 6DAG 0 0 0 1 0 0 0 0 0 2 1 5 FFA 0 0 0 1 0 1 1 0 0 4 3 11 TAG 1 2 4 11 12 2 1 1 45 7 77 Sum 3 3 4 14 1 3 3 1 2 53 12 100 Residual Biomass 5 4 314 1 3 4 1 3 47 13 100 PL 3 2 1 5 0 0 1 0 1 10 4 28 DAG 0 0 0 0 0 0 0 00 1 0 2 FFA 0 0 0 1 0 1 0 0 0 7 2 13 TAG 1 2 3 8 1 1 2 1 1 31 6 57 Sum 44 4 15 1 3 3 1 3 49 12 100 Oil 2 2 4 14 1 3 3 1 2 56 11 100 PL 0 0 0 0 00 0 0 0 0 0 0.7 DAG 0 0 0 1 0 0 0 0 0 2 1 5 FFA 0 0 0 0 0 0 0 0 0 4 1 8TAG 1 2 4 13 1 2 2 1 2 50 8 86 Sum 2 3 5 14 1 2 3 1 2 57 10 100

Example 20 Extraction of Residual Phospholipids with SCF CO₂/EtOH

The purpose of this Example was to demonstrate extraction of a firstresidual biomass sample with a mixture of supercritical CO₂ and ethanolas the extractant to obtain a PL fraction and a second residual biomasssample.

An 18-mL extraction vessel fabricated from 316 SS tubing (1.27 cmo.d.×0.94 cm i.d.×26.0 cm long) was charged with 6.39 g of residualbiomass (extracted Yarrowia lipolytica strain Y8672) from Example 13,which is referred to here as the first residual biomass. The materialwas flushed with CO₂, and then pressurized to 222 bar with a CO₂/ethanolmixture (the extractant) at 40° C. The CO₂ flow rate was 2.3 g/min andthe ethanol flow rate was 0.12 g/min, giving an ethanol concentration of5.0 wt % in the solvent fed to the extraction vessel. The first residualbiomass was extracted at these conditions for 5.3 hr, giving a finalsolvent-to-feed ratio of 120 g CO₂/ethanol per g residual biomass. Theextraction yield of oil was 2.4 wt % from this previously-extractedmaterial. The Table below summarizes lipid analyses for the firstresidual biomass (the starting sample for this Example), the secondresidual biomass (the first residual biomass after extraction in thisExample), and the oil obtained by extraction of the first residualbiomass. As can be seen from the data in the Table below, the oil wasfound to comprise essentially pure PLs. The extractions performedpreviously in Example 13 had already removed neutral lipids and freefatty acids from the yeast cells.

TABLE 22 Example 20: Weight Percent Distribution of Lipid Components18:3 20:3 20:4 16:0 18:0 18:1 18:2 (n-3) 20:2 (n-6) 20:4 (n-3) 20:5Sample palmitic Stearic Oleic Linoleic ALA EDA HGLA ARA ETA EPA otherTotal First Residual 6 3 4 15 1 3 2 1 3 43 15 100 Biomass PL 5 2 1 9 0 01 0 1 12 5 37 DAG 0 0 0 1 0 0 0 0 0 2 1 5 FFA 1 1 0 1 0 1 0 0 0 8 2 15TAG 1 1 2 7 1 1 1 0 1 24 4 42 Sum 7 4 4 17 1 2 2 1 2 46 11 100 SecondResidual Biomass 6 3 4 16 1 2 2 1 4 48 8 100 PL 5 2 1 8 0 0 1 0 1 10 535 DAG 0 0 0 1 0 0 0 0 0 2 1 5 FFA 1 1 0 1 0 1 0 0 0 7 2 14 TAG 1 1 2 71 1 1 0 1 26 4 45 Sum 7 4 4 17 1 2 2 1 2 45 12 100 Oil Extracted 8 5 415 1 3 3 1 2 43 14 100 from First Residual Biomass (PL)

Example 21

The purpose of this Example is to provide alternative microbial biomasscomprising at least one polyunsaturated fatty acid that could beutilized as microbial biomass in the pelletization, extraction,fractionation and distillation methods described herein.

Although numerous oleaginous yeast genetically engineered for productionof omega-3/omega-6 PUFAs are suitable microbial biomass according to theinvention described in the present application, representative strainsof the oleaginous yeast Yarrowia lipolytica are described in Table 5.These include the following strains that have been deposited with theATCC: Y. lipolytica strain Y2047 (producing ARA; ATCC Accession No.PTA-7186); Y. lipolytica strain Y2096 (producing EPA; ATCC Accession No.PTA-7184); Y. lipolytica strain Y2201 (producing EPA; ATCC Accession No.PTA-7185); Y. lipolytica strain Y3000 (producing DHA; ATCC Accession No.PTA-7187); Y. lipolytica strain Y4128 (producing EPA; ATCC Accession No.PTA-8614); Y. lipolytica strain Y4127 (producing EPA; ATCC Accession No.PTA-8802); Y. lipolytica strain Y8406 (producing EPA; ATCC Accession No.PTA-10025); Y. lipolytica strain Y8412 (producing EPA; ATCC AccessionNo. PTA-10026); and Y. lipolytica strain Y8259 (producing EPA; ATCCAccession No. PTA-10027).

Thus, for example, Table 5 shows microbial hosts producing from 25.9% to34% GLA of total fatty acids, from 10.9% to 14% ARA of total fattyacids, from 9% to 61.8% EPA of total fatty acids and 5.6% DHA of totalfatty acids.

One of skill in the art will appreciate that the methodology of thepresent invention is not limited to microbial biomass demonstratinghigh-level EPA production but is equally suitable to microbial biomassdemonstrating high-level production of alternate omega-3/omega-6 PUFAsor combinations or PUFAs thereof.

Example 22A Preparation of Untreated Microbial Biomass Comprising EPAfrom Yarrowia lipolytica Strain Z1978

This example describes recombinant Yarrowia lipolytica strain Z1978,engineered for the production of EPA, and means used to culture thisstrain using a 2-stage fed-batch process. The microbial biomass waspretreated to result in a dried, untreated microbial biomass, having56.1 EPA % TFAs.

Generation of Yarrowia lipolytica Strain Z1978 from Strain Y9502

The development of strain Z1978 from strain is described in U.S. patentapplication Ser. No. 13/218,591 (E.I. duPont de Nemours & Co., Inc.,Attorney Docket Number CL4783USNA, filed Aug. 26, 2011), herebyincorporated herein by reference.

Specifically, to disrupt the Ura3 gene in strain Y9502 (see MATERIALS,supra), construct pZKUM (FIG. 6A; SEQ ID NO:1; described in Table 15 ofU.S. Pat. Appl. Pub. No. 2009-0093543-A1) was used to integrate an Ura3mutant gene into the Ura3 gene of strain Y9502. Transformation wasperformed according to the methodology of U.S. Pat. Appl. Pub. No.2009-0093543-A1, hereby incorporated herein by reference. A total of 27transformants (selected from a first group comprising 8 transformants, asecond group comprising 8 transformants, and a third group comprising 11transformants) were grown on 5-fluoroorotic acid [“FOA”] plates (FOAplates comprise per liter: 20 g glucose, 6.7 g Yeast Nitrogen base, 75mg uracil, 75 mg uridine and an appropriate amount of FOA (Zymo ResearchCorp., Orange, Calif.), based on FOA activity testing against a range ofconcentrations from 100 mg/L to 1000 mg/L (since variation occurs withineach batch received from the supplier)). Further experiments determinedthat only the third group of transformants possessed a real Ura−phenotype.

For fatty acid [“FA”] analysis, cells were collected by centrifugationand lipids were extracted as described in Bligh, E. G. & Dyer, W. J.(Can. J. Biochem. Physiol., 37:911-917 (1959)). Fatty acid methyl esters[“FAMEs”] were prepared by transesterification of the lipid extract withsodium methoxide (Roughan, G., and Nishida I., Arch Biochem Biophys.,276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia cells (0.5 mL culture)were harvested, washed once in distilled water, and dried under vacuumin a Speed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) and a knownamount of C15:0 triacylglycerol (C15:0 TAG; Cat. No. T-145, Nu-CheckPrep, Elysian, Minn.) was added to the sample, and then the sample wasvortexed and rocked for 30 min at 50° C. After adding 3 drops of 1 MNaCl and 400 μl hexane, the sample was vortexed and spun. The upperlayer was removed and analyzed by GC (supra).

Alternately, a modification of the base-catalysed transesterificationmethod described in Lipid Analysis, William W. Christie, 2003 was usedfor routine analysis of the broth samples from either fermentation orflask samples. Specifically, broth samples were rapidly thawed in roomtemperature water, then weighed (to 0.1 mg) into a tarred 2 mLmicrocentrifuge tube with a 0.22 μm Corning® Costar® Spin-X® centrifugetube filter (Cat. No. 8161). Sample (75-800 μl) was used, depending onthe previously determined DCW. Using an Eppendorf 5430 centrifuge,samples are centrifuged for 5-7 min at 14,000 rpm or as long asnecessary to remove the broth. The filter was removed, liquid wasdrained, and ˜500 μl of deionized water was added to the filter to washthe sample. After centrifugation to remove the water, the filter wasagain removed, the liquid drained and the filter re-inserted. The tubewas then re-inserted into the centrifuge, this time with the top open,for ˜3-5 min to dry. The filter was then cut approximately ½ way up thetube and inserted into a fresh 2 mL round bottom Eppendorf tube (Cat.No. 22 36 335-2).

The filter was pressed to the bottom of the tube with an appropriatetool that only touches the rim of the cut filter container and not thesample or filter material. A known amount of C15:0 TAG (supra) intoluene was added and 500 μl of freshly made 1% sodium methoxide inmethanol solution. The sample pellet was firmly broken up with theappropriate tool and the tubes were closed and placed in a 50° C. heatblock (VWR Cat. No. 12621-088) for 30 min. The tubes were then allowedto cool for at least 5 min. Then, 400 μl of hexane and 500 μl of a 1 MNaCl in water solution were added, the tubes were vortexed for 2×6 secand centrifuged for 1 min. Approximately 150 μl of the top (organic)layer was placed into a GC vial with an insert and analyzed by GC.

FAME peaks recorded via GC analysis were identified by their retentiontimes, when compared to that of known fatty acids, and quantitated bycomparing the FAME peak areas with that of the internal standard (C15:0TAG) of known amount. Thus, the approximate amount (μg) of any fattyacid FAME [“μg FAME”] is calculated according to the formula: (area ofthe FAME peak for the specified fatty acid/area of the standard FAMEpeak)*(μg of the standard C15:0 TAG), while the amount (μg) of any fattyacid [“μg FA”] is calculated according to the formula: (area of the FAMEpeak for the specified fatty acid/area of the standard FAME peak)*(μg ofthe standard C15:0 TAG)*0.9503, since 1 μg of C15:0 TAG is equal to0.9503 μg fatty acids. Note that the 0.9503 conversion factor is anapproximation of the value determined for most fatty acids, which rangebetween 0.95 and 0.96.

The lipid profile, summarizing the amount of each individual fatty acidas a weight percent of TFAs, was determined by dividing the individualFAME peak area by the sum of all FAME peak areas and multiplying by 100.

In this way, GC analyses showed that there were 28.5%, 28.5%, 27.4%,28.6%, 29.2%, 30.3% and 29.6% EPA of TFAs in pZKUM-transformants #1, #3,#6, #7, #8, #10 and #11 of group 3, respectively. These seven strainswere designated as strains Y9502U12, Y9502U14, Y9502U17, Y9502U18,Y9502U19, Y9502U21 and Y9502U22, respectively (collectively, Y9502U).

Construct pZKL3-9DP9N (FIG. 6B; SEQ ID NO:2) was then generated tointegrate one delta-9 desaturase gene, one choline-phosphatecytidylyl-transferase gene, and one delta-9 elongase mutant gene intothe Yarrowia YALI0F32131p locus (GenBank Accession No. XM_(—)506121) ofstrain Y9502U. The pZKL3-9DP9N plasmid contained the followingcomponents:

TABLE 23 Description of Plasmid pZKL3-9DP9N (SEQ ID NO: 2) RE Sites AndNucleotides Within SEQ ID NO: 2 Description Of Fragment And ChimericGene Components AscI/BsiWI 884 bp 5′ portion of YALI0F32131p locus(GenBank Accession (887-4) No. XM_506121, labeled as “Lip3-5” in Figure)PacI/SphI 801 bp 3′ portion of YALI0F32131p locus (GenBank Accession(4396-3596) No. XM_506121, labeled as “Lip3-3” in Figure) SwaI/BsiWIYAT1::EgD9eS-L35G::Pex20, comprising: (11716-1) YAT1: Yarrowialipolytica YAT1 promoter (labeled as “YAT” in Figure; U.S. Pat. Appl.Pub. No. 2010-0068789A1); EgD9eS-L35G: Synthetic mutant of delta-9elongase gene (SEQ ID NO: 3; U.S Patent Application No. 13/218,591),derived from Euglena gracilis (“EgD9eS”; U.S. Pat. No. 7,645,604);Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBankAccession No. AF054613) PmeI/SwaI GPDIN::YID9::Lip1, comprising:(8759-11716) GPDIN: Yarrowia lipolytica GPDIN promoter (U.S. Pat. No.7,459,546); YID9: Yarrowia lipolytica delta-9 desaturase gene (GenBankAccession No. XM_501496; SEQ ID NO: 5); Lip1: Lip1 terminator sequencefrom Yarrowia Lip1 gene (GenBank Accession No. Z50020) ClaII/PmeIEXP1::YIPCT::Pex16, comprising: (6501-8759) EXP1: Yarrowia lipolyticaexport protein (EXP1) promoter (labeled as “Exp” in Figure; U.S. Pat.No. 7,932,077); YIPCT: Yarrowia lipolytica choline-phosphate cytidylyl-transferase [“PCT”] gene (GenBank Accession No. XM_502978; SEQ ID NO:7); Pex16: Pex16 terminator sequence from Yarrowia Pex16 gene (GenBankAccession No. U75433) SalI/EcoRI Yarrowia Ura3 gene (GenBank AccessionNo. AJ306421) (6501-4432)

The pZKL3-9DP9N plasmid was digested with AscI/SphI, and then used fortransformation of strain Y9502U17. The transformant cells were platedonto Minimal Media [“MM”] plates and maintained at 30° C. for 3 to 4days (Minimal Media comprises per liter: 20 g glucose, 1.7 g yeastnitrogen base without amino acids, 1.0 g proline, and pH 6.1 (do notneed to adjust)). Single colonies were re-streaked onto MM plates, andthen inoculated into liquid MM at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, resuspended in HighGlucose Media [“HGM”] and then shaken at 250 rpm/min for 5 days (HighGlucose Media comprises per liter: 80 glucose, 2.58 g KH₂PO₄ and 5.36 gK₂HPO₄, pH 7.5 (do not need to adjust)). The cells were subjected tofatty acid analysis, supra.

GC analyses showed that most of the selected 96 strains of Y9502U17 withpZKL3-9DP9N produced 50-56% EPA of TFAs. Five strains (i.e., #31, #32,#35, #70 and #80) that produced about 59.0%, 56.6%, 58.9%, 56.5%, and57.6% EPA of TFAs were designated as Z1977, Z1978, Z1979, Z1980 andZ1981 respectively.

The final genotype of these pZKL3-9DP9N transformant strains withrespect to wildtype Yarrowia lipolytica ATCC #20362 was Ura+, Pex3−,unknown 1−, unknown 2−, unknown 3−, unknown 4−, unknown 5−, unknown6−,unknown 7−, unknown 8−, unknown9−, unknown 10−, unknown 11−,YAT1::ME3S::Pex16, GPD::ME3S::Pex20, YAT1::ME3S::Lip1,FBAINm::EgD9eS::Lip2, EXP1::EgD9eS::Lip1, GPAT::EgD9e::Lip2,YAT1::EgD9eS::Lip2, YAT::EgD9eS-L35G::Pex20, FBAINm::EgD8M::Pex20,EXP1::EgD8M::Pex16, FBAIN::EgD8M::Lip1, GPD::EaD8S::Pex16 (2 copies),YAT1::E389D9eS/EgD8M::Lip1, YAT1::EgD9eS/EgD8M::Aco,FBAINm::EaD9eS/EaD8S::Lip2, GPDIN::YID9::Lip1, GPD::FmD12::Pex20,YAT1::FmD12::Oct, EXP1::FmD12S::Aco, GPDIN::FmD12::Pex16,EXP1::EgD5M::Pex16, FBAIN::EgD5SM::Pex20, GPDIN::EgD5SM::Aco,GPM::EgD5SM::Oct, EXP1::EgD5SM::Lip1, YAT1::EaD5SM::Oct,FBAINm::PaD17::Aco, EXP1::PaD17::Pex16, YAT1::PaD17S::Lip1,YAT1::YICPT::Aco, YAT1::MCS::Lip1, FBA::MCS::Lip1,YAT1::MaLPAAT1S::Pex16, EXP1::YIPCT::Pex16.

Knockout of the YALI0F32131p locus (GenBank Accession No. XM_(—)50612)in strains Z1977, Z1978, Z1979, Z1980 and Z1981 was not confirmed in anyof these EPA strains produced by transformation with pZKL3-9DP9N.

Cells from YPD plates of strains Z1977, Z1978, Z1979, Z1980 and Z1981were grown and analyzed for total lipid content and composition,according to the methodology below.

For a detailed analysis of the total lipid content and composition in aparticular strain of Y. lipolytica, flask assays were conducted asfollowed. Specifically, one loop of freshly streaked cells wasinoculated into 3 mL Fermentation Medium [“FM”] medium and grownovernight at 250 rpm and 30° C. (Fermentation Medium comprises perliter: 6.70 g/L yeast nitrogen base, 6.00 g KH₂PO₄, 2.00 g K₂HPO₄, 1.50g MgSO₄*7H₂O, 20 g glucose and 5.00 g yeast extract (BBL)). TheOD_(600 nm) was measured and an aliquot of the cells was added to afinal OD_(600 nm) of 0.3 in 25 mL FM medium in a 125 mL flask. After 2days in a shaker incubator at 250 rpm and at 30° C., 6 mL of the culturewas harvested by centrifugation and resuspended in 25 mL HGM in a 125 mLflask. After 5 days in a shaker incubator at 250 rpm and at 30° C., a 1mL aliquot was used for fatty acid analysis (supra) and 10 mL dried fordry cell weight [“DCW”] determination.

For DCW determination, 10 mL culture was harvested by centrifugation for5 min at 4000 rpm in a Beckman GH-3.8 rotor in a Beckman GS-6Rcentrifuge. The pellet was resuspended in 25 mL of water andre-harvested as above. The washed pellet was re-suspended in 20 mL ofwater and transferred to a pre-weighed aluminum pan. The cell suspensionwas dried overnight in a vacuum oven at 80° C. The weight of the cellswas determined.

Total lipid content of cells [“TFAs % DCW”] is calculated and consideredin conjunction with data tabulating the concentration of each fatty acidas a weight percent of TFAs [“% TFAs”] and the EPA content as a percentof the dry cell weight [“EPA % DCW”].

Thus, Table 24 below summarizes total lipid content and composition ofstrains Z1977, Z1978, Z1979, Z1980 and Z1981, as determined by flaskassays. Specifically, the Table summarizes the total dry cell weight ofthe cells [“DCW”], the total lipid content of cells [“TFAs % DCW”], theconcentration of each fatty acid as a weight percent of TFAs [“% TFAs”]and the EPA content as a percent of the dry cell weight [“EPA % DCW”].

TABLE 24 Total Lipid Content And Composition In Yarrowia Strains Z1977,Z1978, Z1979, Z1980 and Z1981 By Flask Assay EPA DCW TFAs % % TFAs %Strain (g/L) DCW 16:0 16:1 18:0 18:1 18:2 ALA EDA DGLA ARA EtrA ETA EPAother DCW Z1977 3.8 34.3 2.0 0.5 1.9 4.6 11.2 0.7 3.1 3.3 0.9 0.7 2.259.1 9.9 20.3 Z1978 3.9 38.3 2.4 0.4 2.4 4.8 11.1 0.7 3.2 3.3 0.8 0.62.1 58.7 9.5 22.5 Z1979 3.7 33.7 2.3 0.4 2.4 4.1 10.5 0.6 3.2 3.6 0.90.6 2.2 59.4 9.8 20.0 Z1980 3.6 32.7 2.1 0.4 2.2 4.0 10.8 0.6 3.1 3.50.9 0.7 2.2 59.5 10.0 19.5 Z1981 3.5 34.3 2.2 0.4 2.1 4.2 10.6 0.6 3.33.4 1.0 0.8 2.2 58.5 10.7 20.1

Strain Z1978 was subsequently subjected to partial genome sequencing(U.S. patent application Ser. No. 13/218,591). This work determined thatfour (not six) delta-5 desaturase genes were integrated into theYarrowia genome (i.e., EXP1::EgD5M::Pex16, FBAIN::EgD5SM::Pex20,EXP1::EgD5SM::Lip1, and YAT1::EaD5SM::Oct).

Fermentation of Yarrowia lipolytica Strain Z1978

Yarrowia lipolytica strain Z1978 was grown in a 2-stage fed-batchprocess, as described in the MATERIALS section, supra.

After fermentation, the yeast biomass was dewatered and washed to removesalts and residual medium, and to minimize lipase activity. Drum dryingfollowed, to reduce the moisture to less than 5% to ensure oil stabilityduring short term storage and transportation.

Characterization of the Dried and Untreated Yarrowia lipolytica StrainZ1978 Biomass

The fatty acid composition of the dried and untreated yeast biomass wasanalyzed using the following gas chromatography [“GC”] method.Specifically, the triglycerides were converted to fatty acid methylesters [“FAMEs”] by transesterification using sodium methoxide inmethanol. The resulting FAMEs were analyzed using an Agilent 7890 GCfitted with a 30-m×0.25 mm (i.d.) OMEGAWAX (Supelco) column afterdilution in toluene/hexane (2:3). The oven temperature was increasedfrom 160° C. to 200° C. at 5° C./min, and then 200° C. to 250° C. (holdfor 10 min) at 10° C./min.

FAME peaks recorded via GC analysis were identified by their retentiontimes, when compared to that of known methyl esters [“MEs”], andquantitated by comparing the FAME peak areas with that of the internalstandard (C15:0 triglyceride, taken through the transesterificationprocedure with the sample) of known amount. Thus, the approximate amount(mg) of any fatty acid FAME [“mg FAME”] is calculated according to theformula: (area of the FAME peak for the specified fatty acid/area of the15:0 FAME peak)*(mg of the internal standard C15:0 FAME). The FAMEresult can then be corrected to mg of the corresponding fatty acid bydividing by the appropriate molecular weight conversion factor of1.042-1.052.

The lipid profile, summarizing the amount of each individual fatty acidas a weight percent of TFAs, was approximated (to within ±0.1 weight %)by dividing the individual FAME peak area by the sum of all FAME peakareas and multiplying by 100.

The dried and untreated yeast biomass from Yarrowia lipolytica strainZ1978 contained 56.1 EPA % TFAs, as shown in the Table below.

TABLE 25 Fatty Acid Composition Of Dried And Untreated Z1978 BiomassFatty acid Weight Percent Of Total Fatty Acids C18:2 (omega-6) 14.2C20:5 EPA 56.1 C22:6 DHA non-detectable (<0.05) Other components 29.7

Example 22B Preparation of a SPD-Purified Microbial Oil Having ReducedSterol Content from Untreated Yarrowia lipolytica Strain Z1978 Biomass

The present Example describes means used to disrupt the dried anduntreated Yarrowia lipolytica strain Z1978 biomass of Example 22A viaextrusion and pelletization, extract the oil using supercritical fluidextraction [“SCFE”], and reduce the sterol content of the oil bydistillation, using short path distillation conditions to result in alipid-containing fraction (i.e., the SPD-purified microbial oil).

Disruption and Pelletization Via Extrusion of Dried, Untreated YeastBiomass

The dried and untreated Y. lipolytica strain Z1978 biomass of Example22A was fed to a twin screw extruder. Specifically, a mixture of 84weight percent yeast (containing approximately 39% total microbial oil)and 16% diatomaceous earth (Celatom MN-4; EP Minerals, LLC, Reno, Nev.)was fed to a 40 mm twin screw extruder (Coperion Werner PfleidererZSK-40 mm MC, Stuttgart, Germany) at a rate of 23 kg/hr. A water/sucrosesolution made of 26.5% sucrose was injected after the disruption zone ofthe extruder at a flow rate of 70 mL/min. The extruder was operated witha 37 kW motor and high torque shaft, at 140 rpm. The % torque range was17-22. The resulting disrupted yeast powder was cooled to 35° C. in afinal water cooled barrel. The moist extruded powder was fed into a LCIMulti-Granulator Model No. MG-55 (LCI Corporation, Charlotte, N.C.)assembled with a 1 mm hole diameter by 1 mm thick screen and set to 80RPM. Extrudate was formed at 27 kg/hr with a steady 2.2 amp current drawand was dried using conventional drying equipment. Dried pellets,approximately 1 mm diameter×6 to 10 mm in length, had a final moisturecontent of 1.7%, as measured on a Sartorius MA35 moisture analyzer(Sartorius AG, Goettingen, Germany).

Extraction of the Extruded Yeast Biomass

The extruded yeast pellets were extracted using supercritical fluidphase carbon dioxide (CO₂) as the extraction solvent to produce atriglyceride-rich extracted oil containing EPA. Specifically, the yeastpellets were charged to a 320 L stainless steel extraction vessel andpacked between plugs of polyester foam filtration matting (Aero-FloIndustries, Kingsbury, Ind.). The vessel was sealed, and then CO₂ wasmetered by a commercial compressor (Pressure Products Industries)through a heat exchanger (pre-heater) and fed into the verticalextraction vessel to extract the triglyceride-rich oil from the pelletsof disrupted yeast. The extraction temperature was controlled by thepre-heater, and the extraction pressure was maintained with an automatedcontrol valve (Kammer) located between the extraction vessel and aseparator vessel. The CO₂ and oil extract were expanded to a lowerpressure through this control valve. The extracted oil was collectedfrom the expanded solution as a precipitate in the separator. Thetemperature of the expanded CO₂ phase in the separator was controlled byuse of an additional heat exchanger located upstream of the separator.This lower pressure CO₂ stream exited the top of the separator vesseland was recycled back to the compressor through a filter, a condenser,and a mass flow meter. The extracted oil was periodically drained fromthe separator and collected as product.

The extraction vessel was initially charged with 150 kg of the extrudedyeast pellets. The triglyceride-rich oil was then extracted from thepellets with supercritical fluid CO₂ at 5000 psig (345 bar), 55° C., anda solvent-to-feed ratio of 32 kg CO₂ per kg of starting yeast pellets. Atotal of 39.6 kg of extracted oil was collected from the separatorvessel, to which was added about 1000 ppm each of two antioxidants:Covi-ox T70 (Cognis, Ontario, Canada) and Dadex RM (Nealanders, Ontario,Canada). The extracted oil contained 661 mg ergosterol/100 g of oil, asdetermined by GC analysis (infra).

Specifically, ergosterol content was determined by high-performanceliquid chromatography (HPLC) with ultraviolet (UV) detection. Extractedoil samples (100 mg) were diluted with 14 mL of 9:102-propanol:1-heptanol and mixed well. Calibration standards of 96% pureergosterol (Alfa Aesar, Inc., Ward Hill, Mass.) were prepared in therange of 10 to 300 μg/mL in 2-propanol. Samples and standards werechromatographed on a XDB-C8 HPLC column (4.6 mm id., 150 mm length, 5 μmparticle size, Agilent Technologies, Inc., Wilmington, Del.) using an0.02% ammonium carbonate in water—acetonitrile gradient from 65 to 100%acetonitrile in 12.5 min. The injection volume was 5 μL, the flow ratewas 1.2 mL/min and the column temperature was 50° C. The UV (282 nm)response of the ergosterol peak was compared with those of thecalibration standards analyzed under the same conditions.

Distillation Under SPD Conditions

The extracted oil was degassed and then passed through a 6″ stainlesssteel molecular still (POPE Scientific, Saukville, Wis.) using a feedrate of 12 kg/hr to remove residual water. The surface temperatures ofthe evaporator and condenser were set at 140° C. and 15° C.,respectively. The vacuum was maintained at 15 torr. Approximately 3 wt.% of the extracted oil was removed as water in the distillate. Thedewatered, extracted oil was substantially free of phospholipids,containing 0.5 ppm of phosphorous. Upon visual inspection, thedewatered, extracted oil was cloudy at room temperature.

The dewatered, extracted oil was passed through the 6″ molecular stillat a feed rate of 12 kg/hr for a second time. The vacuum was lowered to1 mtorr, and the surface temperatures of the evaporator and condenserwere maintained at 240° C. and 50° C., respectively. Approximately 7 wt.% of the dewatered, extracted oil was removed as the distillate; thisfraction contained mainly free fatty acids and ergosterol. Atriacylglycerol-containing fraction (i.e., the lipid-containing fractionor SPD-purified oil) was also obtained, containing 284 mg ergosterol/100g oil (a ˜57% reduction in ergosterol content, when compared toergosterol content in the extracted oil). The SPD-purified oil was clearafter being stored at 10° C. for several days.

Example 23 Preparation of a SPD-Purified Microbial Oil Having ReducedSterol Content from Untreated Yarrowia lipolytica Strain Y9502 Biomass

The present Example describes means used to disrupt dried and untreatedYarrowia lipolytica strain Y9502 biomass via extrusion, extract the oilusing supercritical fluid extraction [“SCFE”], and reduce the sterolcontent of the oil by distillation, using short path distillationconditions to result in a lipid-containing fraction (i.e., theSPD-purified microbial oil).

Preparation of Dried and Untreated Yarrowia lipolytica Strain Y9502Biomass

Y. lipolytica strain Y9502 was cultured in a 2-stage fed-batch processand the resulting microbial biomass was dewatered, washed and dried,according to the methodology set forth in Example 22A.

Disruption Via Extrusion of Dried, Untreated Yeast Biomass

The dried and untreated Y. lipolytica strain Y9502 biomass was fed to atwin screw extruder. Specifically, the yeast biomass (containingapproximately 37% total microbial oil) was fed to a 70 mm twin screwextruder (Coperion Werner Pfleiderer ZSK-70 mm SCD, Stuttgart, Germany)at a rate of 270 kg/hr, in the absence of diatomaceous earth.

The extruder was operated with a 150 kW motor and high torque shaft at150 rpm and 33 percent of the total amp range. The resulting disruptedyeast biomass was cooled to 81° C. in the final water cooled barrel. Themoisture content of the disrupted biomass was 2.8 wt. ° A), as measuredon a Sartorius MA35 moisture analyzer (Sartorius AG, Goettingen,Germany).

Extraction of the Extruded Yeast Biomass

The extruded yeast biomass was mixed with diatomaceous earth to preventbed compaction and extracted using supercritical fluid phase CO₂ as theextraction solvent to produce a crude triglyceride oil containing EPA(i.e., “Extracted oil”). Specifically, a total of 82.7 kg of theextruded yeast biomass was mixed with 41 kg of diatomaceous earth(Celatom MN-4; EP Minerals, LLC, Reno, Nev.) and charged to a 320 Lstainless steel extraction vessel, configured in a manner identical tothat described in Example 22B, with the following exceptions: (i) theextraction temperature was controlled to 40° C. by the pre-heater; (ii)the extraction pressure was maintained at 4500 psig (310 bar); (iii) asolvent-to-feed ratio of 44 kg CO₂ per kg of starting yeast was used forthe extraction. In this way, 23.2 kg oil was extracted from thedisrupted yeast. The extracted oil contained 774 mg ergosterol/100 goil, as determined by GC analysis according to the methodology ofExample 22B.

Distillation Under SPD Conditions

The extracted oil was passed through a 2″ glass molecular still toprovide a dewatered, extracted oil. The flow rate was maintained atapproximately 480 g/hr. The vacuum, evaporator and condensertemperatures were 0.2 mm Hg, 130° C. and 60° C., respectively. Thedewatered, extracted oil was then passed through the still three timesat different temperatures at a vacuum of 1 mtorr, as shown in the Tablebelow. After each pass, the ergosterol level, EPA content (as a wt. % ofTFAs) and total Omega-3 content (as a wt. % of TFAs) in thetriacylglycerol-containing fraction (i.e., the lipid-containing fractionor SPD-purified oil) were determined, as previously described.

TABLE 26 Ergosterol And PUFA Content In SPD-Purified Oil Pass 1 Pass 2Pass 3 Temperature (° C.) 210 240 270 Ergosterol (mg/100 g) 110 52.81.21 C20:5 EPA (wt. % TFAs) 54.9 55.2 55.4 Total Omega-3 (wt. % TFAs)57.51 57.92 57.18Thus, at 210° C., the ergosterol level in the SPD-purified oil was 110mg/100 g of oil and it was reduced to about 53 mg/100 g of oil at 240°C. The ergosterol was almost completely removed to 1 mg/100 g of oilwhen the temperature was further increased to 270° C. This correspondsto a ˜57%, ˜86% and ˜99.8% reduction in ergosterol content in Pass 1,Pass 2 and Pass 3, respectively, when compared to ergosterol content inthe extracted oil.

With respect to the PUFA content in the SPD-purified oil, the data ofTable 26 demonstrate that no significant degradation of EPA or totalOmega-3 content occurred, even when the oil was passed through the SPDstill at 270° C.

The SPD-purified oil of Pass 3 was further analyzed for the appearanceof unexpected components and contaminants using chromatographicprofiling. Specifically, testing was done by: (i) gas chromatographywith flame ionization detection (GC/FID); (ii) thin-layer chromatography(TLC); and, (iii) liquid chromatography with mass spectrometric, lightscattering and ultraviolet detection (HPLC/MS/ELSD/UV). The GC/FIDprofile was run on the methyl esters of the SPD-purified oil sample. TheTLC and HPLC/MS/ELSD/UV profiles were run on the SPD-purified oildirectly. In all cases, the SPD-purified oil profile was compared with areference oil prepared with Y. lipolytica strain Y4305 biomass(MATERIALS, supra).

Specifically, the reference oil was produced from dried and untreated Y.lipolytica strain Y4305 biomass, according to the methodology set forthin Example 22A. The dried and untreated biomass was mechanicallydisrupted using a media mill with an oil to iso-hexane solvent ratio of1 to 7. The residual biomass (i.e., cell debris) was removed using adecanter centrifuge and the solvent was evaporated to yield an extractedoil containing triglycerides. The extracted oil was degummed using coldacetone with an extracted oil to solvent ratio of 1 to 1.5, followed byacid degumming with 50% aqueous citric acid. The degummed oil was thenbleached with an acid-activated clay and deodorized at 210° C. for 30min to yield the reference oil sample.

None of the chromatographic profiles of the SPD-purified oil of Pass 3contained any peaks that were not seen in the profile of the referencesample. Both samples were run on the same day under the same conditions.Additionally, there were no unidentified peaks in of the SPD-purifiedoil that had significantly higher responses than the corresponding peaksin the profile of the reference sample. Also, none of the peaks in theSPD-purified oil of Pass 3 had higher responses than the correspondingpeaks in the SPD-purified oil of Pass 1 or Pass 2, which were producedat lower temperatures (i.e., 210° C. and 240° C., respectively). Theseanalyses show that the removal of ergosterol at high temperatures usingSPD does not lead to the appearance of degradation products in the oil;thus, it is hypothesized that no significant degradation of the PUFAsoccurs by application of this processing technique.

Example 24 Preparation of a SPD-Purified Microbial Oil Having ReducedSterol Content from Untreated Yarrowia lipolytica Strain Y8672 Biomass

The present Example describes means used to disrupt dried and untreatedYarrowia lipolytica strain Y8672 biomass via mechanical disruption usinga media mill, extract the crude oil using iso-hexane solvent, and reducethe sterol content of the acetone-degummed oil by distillation, usingshort path distillation conditions to result in a lipid-containingfraction (i.e., the SPD-purified microbial oil).

Preparation of Dried and Untreated Yarrowia lipolytica Strain Y8672Biomass

Y. lipolytica strain Y8672 was cultured in a 2-stage fed-batch processand the resulting microbial biomass was dewatered, washed and dried,according to the methodology set forth in Example 22A.

Disruption and Extraction Via Media Mill and Iso-Hexane Solvent ofDried, Untreated Yeast Biomass to Produce Extracted Oil

The dried and untreated Y. lipolytica strain Y8672 biomass wasmechanically disrupted using a media mill with iso-hexane solvent. Theresidual biomass (i.e., cell debris) was removed using a decantercentrifuge and the solvent was evaporated to yield an extracted oilcontaining triglycerides.

The extracted oil was analyzed using the methodology of Example 22B. Themicrobial oil contained 58.1 EPA % TFAs, as shown in the Table below.

TABLE 27 Fatty Acid Composition of Extracted Y8672 Microbial Oil Fattyacid Weight Percent Of Total Fatty Acids C18:2 (omega-6) 15.6 C20:5 EPA58.1 C22:6 DHA non-detectable Other components 26.3

A portion of the extracted oil was degummed using cold acetone with aextracted oil to solvent ratio of 1 to 1.5. The acetone-degummed oilcontained 880 mg ergosterol/100 g oil and 74.5 ppm of phosphorous.

Distillation Under SPD Conditions

The acetone-degummed oil was subjected to short path distillation,according to the methodology of Example 22B (except the evaporatortemperature was set at 255° C.). Almost no distillate was collectedduring the first pass since there was very little water in theacetone-degummed oil. During the second pass, roughly 12 wt. % ofdistillate was collected. The final ergosterol level in thetriacylglycerol-containing fraction (i.e., the lipid-containing fractionor SPD-purified oil) was 106 mg/100 g (a ˜88% reduction in ergosterolcontent, when compared to ergosterol content in the acetone-degummedoil); the SPD-purified oil contained 66 ppm of phosphorous.

Example 25 Preparation of a Non-Concentrated Microbial Oil Comprising56.1% EPA of Total Fatty Acids [“TFAs”]

The present Example describes the isolation of a non-concentratedmicrobial oil obtained from microbial biomass of recombinant Yarrowialipolytica strain Z1978 cells, engineered for the production of EPA.

Specifically, Y. lipolytica strain Z1978 was cultured using a 2-stagefed-batch process. Microbial oil was then isolated from the biomass viadrying, extracted (via a combination of extrusion, pelletization andsupercritical fluid extraction), and purified via short pathdistillation, yielding a non-concentrated, triglyceride-richSPD-purified oil comprising 56.1 EPA % TFAs (i.e., the lipid-containingfraction).

Fermentation and Disruption Via Extrusion and Pelletization of Dried,Untreated Yarrowia lipolytica Strain Z1978 Biomass

A Y. lipolytica strain Z1978 culture was fermented and the microbialbiomass was harvested and dried, as described in the MATERIALS, supra.The dried and untreated biomass was then fed to a twin screw extruder.Specifically, a mixture of the biomass and 15% of diatomaceous earth(Celatom MN-4 or Celite 209, EP Minerals, LLC, Reno, Nev.) were premixedand then fed to ZSK-40 mm MC twin screw extruder (Coperion Werner &Pfleiderer, Stuttgart, Germany) at a rate of 45.5 kg/hr. A water/sucrosesolution made of 26.5% sucrose was injected after the disruption zone ofthe extruder at a flow rate of 147 mL/min. The extruder was operated at280 rpm with a % torque range of 20-23. The resulting disrupted yeastpowder was cooled to 35° C. in a final water cooled barrel. The moistextruded powder was then fed into a LCI Dome Granulator Model No. TDG-80(LCI Corporation, Charlotte, N.C.) assembled with a multi-bore dome die1 mm diameter by 1 mm thick screen and set to 82 RPM. Extrudate wasformed at 455-600 kg/hr (as-dried rate). The sample was dried in avibratory fluid bed dryer (FBP-75, Carman Industries, Inc.,Jeffersonville, Ind.) with a drying zone of 0.50 m² with 1150 standardcubic feet per minute [“scfm”] of air flow maintained at 100° C. and acooling zone of 0.24 m² operating with an air flow estimated at 500-600scfm at 18° C. Dried pellets, approximately 1 mm diameter×6 to 10 mm inlength, exited the dryer in the 25-30° C. range, having a final moisturecontent of 5-6% measured on an O'Haus moisture analyzer (Parsippany,N.J.).

Oil Extraction of the Extruded Yeast Biomass

The extruded yeast pellets were extracted using supercritical fluidphase CO₂ as the extraction solvent to produce non-concentratedtriglyceride-rich extracted oil, using a 320 L stainless steelextraction vessel as described in Example 22B.

The extraction vessel was initially charged with approximately 150 kg ofthe extruded yeast pellets. The non-concentrated extracted oil was thenextracted from the pellets with supercritical fluid CO₂ at 5000 psig(345 bar), 55° C., and a solvent-to-feed ratio ranging from 40 to 50 kgCO₂ per kg of starting yeast pellets. Roughly 37.5 kg ofnon-concentrated extracted oil was collected from the separator vessel,to which was added about 1000 ppm each of two antioxidants, i.e. Covi-oxT70 (Cognis, Mississauga, Canada) and Dadex RM (Nealanders, Mississauga,Canada).

Distillation Under SPD Conditions

The non-concentrated extracted oil was degassed and then passed througha 6″ molecular still (POPE Scientific, Saukville, Wis.) using a feedrate of 12 kg/hr to remove residual water. The surface temperatures ofthe evaporator and condenser were set at 140° C. and 15° C.,respectively. The vacuum was maintained at 15 torr.

The dewatered extracted oil was passed through the molecular still at afeed rate of 12 kg/hr for a second time to remove undesiredlower-molecular weight compounds, such as ergosterol and free fattyacids in the distillate. The vacuum was lowered to 1 mtorr, and thesurface temperatures of the evaporator were maintained between 240° C.and 270° C. A triacylglycerol-containing fraction (i.e., theSPD-purified oil) was obtained, having reduced sterols relative to thesterol content in the non-concentrated extracted oil. Thenon-concentrated SPD-purified oil was cooled to below 40° C. beforepackaging.

Characterization of Non-Concentrated SPD-Purified Oil from Yarrowialipolytica Strain Z1978

The fatty acid composition of the non-concentrated SPD-purified oil(i.e., the lipid containing fraction) from strain Z1978 was analyzed,following transesterification, according to the methodology of Example27. The SPD-purified oil contained 56.1 EPA % TFAs and DHA wasnon-detectable (i.e. <0.05%), as shown below in Table 28.

TABLE 28 Fatty Acid Composition Of Non-Concentrated Yarrowia lipolyticaZ1978 SPD-Purified Oil Fatty acid Weight Percent Of Total Fatty AcidsC18:2 (omega-6) 14.2 C20:5 EPA 56.1 C22:6 DHA non-detectable (<0.05%)Other components 29.7

Example 26 Creation of Solid Pellets from Nannochloropsis Algae and OilExtraction Thereof

The present example describes tests performed to demonstrate theapplicability of the methodologies disclosed herein for use with amicrobial biomass other than Yarrowia. Specifically, Nannochloropsisbiomass was mixed with a grinding agent and binding agent, to providesolid pellets. These pellets were subjected to supercritical CO₂extraction and total extraction yields were compared.

Kuehnle Agrosystems, Inc. (Honolulu, Hi.) provides a variety of axenic,unialgal stock algae for purchase. Upon request, they suggested algaestrain KAS 604, comprising a Nannochloropsis species, as an appropriatemicrobial biomass having a lipid content of at least 20%. The biomasswas grown under standard conditions (not optimizing conditions for oilcontent) and dried by Kuehnle Agrosystems, Inc. and then the microalgaepowder was purchased for use below.

91.7 parts of microalgae powder were premixed in a bag with 8.3 parts ofCelatom MN-4 D-earth. The resultant dry mix was fed at 0.91 kg/hr to an18 mm twin screw extruder (Coperion Werner Pfleiderer ZSK-18 mm MC).Along with the dry feed, a 31% aqueous solution of sugar made of 10.9parts water and 5.0 parts sugar was injected after the disruption zoneof the extruder at a flow-rate of 2.5 mL/min. The extruder was operatingwith a 10 kW motor and high torque shaft, at 200 rpm and % torque rangeof 46-81 to provide a disrupted yeast powder cooled to 31° C. in a finalwater cooled barrel.

The fixable mix was then fed into a MG-55 LCI Dome Granulator assembledwith 1.2 mm diameter holes by 1.2 mm thick screen and set to 20 RPM.Extrudates were formed at 20 kg/hr and a 6-7 amp current. The sample wasdried in a Sherwood Dryer at 70° C. for 20 min to provide solid pelletshaving a final moisture level of 4.9%. The solid pellets, approximately1.2 mm diameter×2 to 8 mm in length, were 82.1% algae, with theremainder of the composition being pelletization aids. The amount oftotal and free oil in the solid Nannochloropsis pellets was thendetermined and compared to the amount of oil extracted from the solidNannochloropsis pellets by SCF.

Determination of Total Oil Content in Solid Nannochloropsis Pellets

Specifically, total oil was determined on the pelletized sample bygently grinding it into a fine powder using a mortar and pestle, andthen weighing aliquots (in triplicate) for analysis. The fatty acids inthe sample (existing primarily as triglycerides) were converted to thecorresponding methyl esters by reaction with acetyl chloride/methanol at80° C. A C15:0 internal standard was added in known amounts to eachsample for calibration purposes. Determination of the individual fattyacids was made by capillary gas chromatography with flame ionizationdetection (GC/FID). The sum of the fatty acids (expressed intriglyceride form) was 6.1%; this was taken to be the total oil contentof the sample. After normalization, since the algae in the pelletsrepresented only 82.1% of the total mass, the total oil content in thealgae was determined to be 7.4% (i.e., 6.1% divided by 0.821).

The distribution of the individual fatty acids within the total oilsample is shown in the Table below.

TABLE 29 Distribution Of Fatty Acids In Solid Nannochloropsis PelletsPercent (w/w) found Fatty Acid (as free fatty acid) Saturated fattyacids 1.4 C16:0 Palmitic acid 1.3 C18:0 Stearic acid 0.06Monounsaturated fatty acids 0.8 C16:1 Palmitoleic acid 0.4 C18:1, n-9Oleic acid 0.2 C18:1 Octadecanoic acid 0.04 Polyunsaturated fatty acids2.7 C18:2, n-6 Linoleic acid 0.8 C18:3, n-3 alpha-Linolenic acid 1.2C20:4, n-6 Arachiodonic acid 0.1 C20:5, n-3 Eicosapentaenoic acid 0.6Unknown fatty acids 1.2

Determination of Free Oil Content in Solid Nannochloropsis Pellets

Free oil is normally determined by stirring a sample with n-heptane,centrifuging, and then evaporating the supernatant to dryness. Theresulting residual oil is then determined gravimetrically and expressedas a weight percentage of the original sample. This procedure was notfound to be satisfactory for the pelletized algae sample, because theresulting residue contained significant levels of pigments. Thus, theprocedure above was modified by collecting the residue as above, addingthe C15:0 internal standard in known amount, and then analyzing byGC/FID using the same parameters as for total oil determination. In thisway, the free oil content of the sample was determined to be 3.7%. Afternormalization, the free oil content in the algae was determined to be4.5% (i.e., 3.7% divided by 0.821).

SCF Extraction of Solid Nannochloropsis Pellets

The extraction vessel was charged with 24.60 g of solid pellets (on adry weight basis), resulting in about 21.24 g of algae on correcting forthe grinding and binding agents. The pellets were flushed with CO₂, thenheated to about 40° C. and pressurized to approximately 311 bar. Thepellets were extracted at these conditions at a flow rate of 3.8 g/minCO₂ for about 6.7 hr, giving a final solvent-to-feed (S/F) ratio ofapproximately 71 g CO₂/g algae. The extraction yield was 6.2% of thecharged algae.

Based on the above, it is concluded that the process described herein[i.e., comprising steps of (a) mixing a microbial biomass, having amoisture level and comprising oil-containing microbes, and at least onegrinding agent capable of absorbing oil, to provide a disrupted biomassmix comprising disrupted microbial biomass; (b) blending at least onebinding agent with said disrupted biomass mix to provide a fixable mixcapable of forming a solid pellet; and (c) forming said solid pelletfrom the fixable mix] can be successfully utilized to produce solidpellets comprising disrupted microbial biomass from Nannochloropsis. Itis hypothesized that the methodology will prove suitable for numerousother oil-containing microbes, although it is expected that optimizationof the process for each particular microbe will lead to increaseddisruption efficiencies.

Furthermore, the present Example demonstrates that the solidNannochloropsis pellets can be extracted with a solvent to provide anextract comprising the oil, in a variety of means. As is well known inthe art, different extraction methods will result in different amountsof extracted oil; it is expected the extraction yields may be increasedfor a particular solid pellet upon optimization of the extractionprocess. Furthermore, it is expected that the extracted oil could besubjected to distillation, under short path distillation conditions,according to the disclosure herein.

Example 27 Preparation of a Microbial Oil Comprising 58.2% EPA of TotalFatty Acids [“TFAs”]

The present Example describes the isolation of a microbial oil obtainedfrom microbial biomass of recombinant Yarrowia lipolytica cells,engineered for the production of EPA. This microbial oil was thenenriched by various means, as described below in Examples 28-30.

Microbial oil was isolated from Y. lipolytica strain Y8672 microbialbiomass via an iso-hexane solvent and purified, yielding anon-concentrated, triglyceride-rich purified oil comprising 58.2 EPA %TFAs.

Fermentation and Extraction of Microbial Oil from Y. lipolytica StrainY8672 Biomass

Y. lipolytica strain Y8672 was grown in 2-stage fed-batch process,dewatered and washed according to the MATERIALS. Drum drying followed toreduce the moisture to less than 5% to ensure oil stability during shortterm storage and transportation of the untreated microbial biomass.

The microbial biomass was then subjected to mechanical disruption withiso-hexane solvent to extract the EPA-rich microbial oil from thebiomass. The residual biomass (i.e., cell debris) was removed and thesolvent was evaporated to yield an extracted oil. The extracted oil wasdegummed using phosphoric acid and refined with 20° Baume caustic toremove phospholipids, trace metals and free fatty acids. Bleaching withsilica and clay was used to adsorb color compounds and minor oxidationproducts. The last deodorization step stripped out volatile, odorous andadditional color compounds to yield a non-concentrated purifiedmicrobial oil comprising PUFAs in their natural triglyceride form.

Characterization of Microbial Oil from Y. lipolytica Strain Y8672

The fatty acid composition of the non-concentrated purified oil wasanalyzed using the GC method set forth in Example 22A.

The results obtained from the GC analyses on the non-concentrated Y8672purified oil are shown below in Table 30. The purified oil contained58.2 EPA % TFAs and DHA was non-detectable (i.e. <0.05%).

TABLE 30 Fatty Acid Composition Of Non-Concentrated Y8672 Purified OilFatty acid Weight Percent Of Total Fatty Acids C18:2 (omega-6) 16.6C20:5 EPA 58.2 C22:6 DHA non-detectable (<0.05%) Other components 25.2One of skill in the art would expect that a microbial oil of similarcomposition could be obtained from Y. lipolytica strain Y8672, if thebiomass was subjected to pelletization, extraction and then distillationunder short path distillation conditions.

Example 28 Enrichment of Microbial Oil Via Urea Adduct Formation

This example demonstrates that an EPA concentrate comprising up to 78%EPA ethyl esters, measured as a weight percent of oil, and substantiallyfree of DHA could be obtained upon enrichment of the non-concentratedpurified oil from Example 27 via urea adduct formation.

KOH (20 g) was first dissolved in 320 g of absolute ethanol. Thesolution was then mixed with 1 kg of the non-concentrated purified oilfrom Example 27 and heated to approximately 60° C. for 4 hrs. Thereaction mixture was left undisturbed in a Sep funnel overnight forcomplete phase separation. After removing the bottom glycerol fraction,a small amount of silica was added to the upper ethyl ester fraction toremove excess soap. The ethanol was rotovapped off at about 90° C. undervacuum, which yielded clear, but light-brown, ethyl esters.

The ethyl esters (20 g) were mixed with 40 g of urea and 100 g ofethanol (90% aqueous) at approximately 65° C. The mixture was maintainedat this temperature until it turned into a clear solution. The mixturewas then cooled to and held at room temperature for approximately 20 hrsfor urea crystals and adducts to form. The solids were then removedthrough filtration and the liquid fraction was rotovapped to removeethanol. The recovered ethyl ester fraction was washed with a first andthen a second wash of 200 mL of warm water. The pH of the solution wasadjusted to 3-4 first before decanting off the aqueous fraction. Theethyl ester fraction was then dried to remove residual water.

To determine the fatty acid ethyl ester [“FAEE”] concentrations in theethyl ester fraction, the FAEEs were analyzed directly after dilution intoluene/hexane (2:3), using the same GC conditions and calculations aspreviously described in Example 22A to determine FAME concentrations.The only modifications in methodology were: i) C23:0 EE was used as theinternal standard instead of C15:0; and, ii) the molecular weightconversion factor of 1.042-1.052 was not required.

EPA ethyl ester [“EPA-EE”], however, was subjected to a slightlymodified procedure from that above. Specifically, a reference EPA-EEstandard of known concentration and purity was prepared to containapproximately the same amount of EPA-EE expected in the analyticalsamples, as well as the same amount of C23:0 EE internal standard. Theexact amount of EPA-EE (mg) in a sample is calculated according to theformula: (area of EPA-EE peak/area of the C23:0 EE peak)×(area of theC23:0 EE peak in the calibration standard/area of the EPA-EE peak in thecalibration standard)×(mg EPA-EE in the calibration standard). Allinternal and reference standards were obtained from Nu-Chek Prep, Inc.

In this way, the FAEE concentrations were determined in the enriched oilfraction, i.e., the EPA concentrate. Specifically, enrichment of thenon-concentrated purified oil via urea adduct formation yielded an EPAconcentrate with 77% EPA ethyl ester, measured as a weight percent ofoil, and substantially free of DHA, as shown in Table 31.

TABLE 31 EPA Ethyl Ester Concentrate With Urea Adduct Method Fatty acidethyl esters Weight Percent Of Oil C18:2 (omega-6)  3.9 C20:5 EPA 76.5C22:6 DHA non-detectable (<0.05%) Other components 19.6

One of ordinary skill in the art will appreciate that the EPAconcentrate, comprising 77% EPA ethyl ester, measured as a weightpercent of oil, and substantially free of DHA, could readily beconverted to yield an EPA concentrate in an alternate form (i.e., theEPA ethyl ester could be converted to free fatty acids,triacylglycerols, methyl esters, and combinations thereof), using meanswell known to those of skill in the art. Thus, for example, the 77% EPAethyl ester could be re-esterified to triglycerides via glycerolysis, toresult in an EPA concentrate, in triglyceride form, comprising at least70 wt % of EPA, measured as a wt % of oil, and substantially free ofDHA.

Example 29 Enrichment of Microbial Oil Via Liquid Chromatography

This example demonstrates that an EPA concentrate comprising up to 95.4%EPA ethyl ester, measured as a weight percent of oil, and substantiallyfree of DHA could be obtained upon enrichment of the non-concentratedpurified oil from Example 27 using a liquid chromatography method.

The non-concentrated purified oil from Example 27 was transesterified toethyl esters using a similar method as described in Example 28 but withsome minor modifications (i.e., use of sodium ethoxide as a basecatalyst instead of potassium hydroxide).

The ethyl esters were then enriched by Equateq (Isle of Lewis, Scotland)using their liquid chromatographic purification technology. Variousdegrees of enrichment were achieved (e.g., see exemplary data for Sample#1 and Sample #2, infra). Thus, enrichment of the non-concentratedpurified oil via liquid chromatography yielded an EPA concentrate withup to 95.4% EPA ethyl ester, measured as a weight percent of oil, andsubstantially free of DHA, as shown in Table 32.

TABLE 32 EPA Ethyl Ester Concentrate With A Liquid ChromatographyEnrichment Method Weight Percent Of Oil Fatty acid ethyl esters Sample#1 Sample #2 C18:2 (omega-6)  5.7 ND C20:5 EPA 82.8 95.4 C22:6 DHAnon-detectable non-detectable (<0.05%) (<0.05%) Other components 11.5 4.6

One of skill in the art will appreciate that the EPA concentrate,comprising either 82.8% EPA ethyl ester or 95.4% EPA ethyl ester,measured as a weight percent of oil, and substantially free of DHA,could readily be converted to yield an EPA concentrate in an alternateform (i.e., the EPA ethyl ester could be converted to free fatty acids,triacylglycerols, methyl esters, and combinations thereof), using meanswell known to those of skill in the art. Thus, for example, the 82.8%EPA ethyl ester or 95.4% EPA ethyl ester could be re-esterified totriglycerides via glycerolysis, to result in an EPA concentrate, intriglyceride form, comprising at least 70 wt % of EPA, measured as a wt% of oil, and substantially free of DHA.

Example 30 Enrichment of Microbial Oil Via Supercritical FluidChromatography

This example demonstrates that an EPA concentrate comprising up to 89.8%EPA ethyl esters, measured as a weight percent of oil, and substantiallyfree of DHA could be obtained upon enrichment of the non-concentratedpurified oil from Example 27 using a supercritical fluid chromatographic[“SFC”] method.

The non-concentrated purified oil from Example 27 was transesterified toethyl esters using sodium ethoxide as a base catalyst, and thenprocessed through an adsorption column to remove compounds that wereinsoluble in supercritical CO₂. The processed ethyl ester oil was thenpurified by K.D. Pharma (Bexbach, Germany) using their supercriticalchromatographic technology. Various degrees of enrichment were achieved(e.g., see exemplary data for Sample #1 and Sample #2, infra). Thus,enrichment of the non-concentrated purified oil via SFC yielded an EPAconcentrate with 85% and 89.8% EPA ethyl esters, measured as a weightpercent of oil, and substantially free of DHA, as shown in Table 33.

TABLE 33 EPA Ethyl Ester Concentrate With SFC Enrichment Method WeightPercent Of Oil Fatty acid ethyl esters Sample #1 Sample #2 C18:2(omega-6) 0.4 0.2 C20:5 EPA 85 89.8 C22:6 DHA Non-detectablenon-detectable (<0.05%) (<0.05%) Other components 14.6 10

One of skill in the art will appreciate that the EPA concentrate,comprising either 85% EPA ethyl ester or 89.8% EPA ethyl ester, measuredas a weight percent of oil, and substantially free of DHA, could readilybe converted to yield an EPA concentrate in an alternate form (i.e., theEPA ethyl ester could be converted to free fatty acids,triacylglycerols, methyl esters, and combinations thereof), using meanswell known to those of skill in the art. Thus, for example, the 85% EPAethyl ester or 89.8% EPA ethyl ester could be re-esterified totriglycerides via glycerolysis, to result in an EPA concentrate, intriglyceride form, comprising at least 70 wt % of EPA, measured as a wt% of oil, and substantially free of DHA.

Example 31 Preparation of a Microbial Oil Comprising 56.1% EPA of TotalFatty Acids [“TFAs”]

The present Example describes the isolation of a microbial oil obtainedfrom microbial biomass of recombinant Yarrowia lipolytica cells,engineered for the production of EPA. This microbial oil was thenenriched by fractional distillation, as described infra in Example 32.

Specifically, Y. lipolytica strain Z1978 was recombinantly engineered toenable production of about 58.7 EPA % TFAs and cultured using a 2-stagefed-batch process. Microbial oil was then isolated from the biomass viadrying, extracted (via a combination of extrusion, pelletization andsupercritical fluid extraction), and purified via short pathdistillation, yielding a non-concentrated, triglyceride-richSPD-purified oil (i.e., a lipid-containing fraction) comprising 56.1 EPA% TFAs.

Fermentation and Disruption Via Extrusion and Pelletization of Dried,Untreated Y. lipolytica Strain Z1978 Biomass

A Y. lipolytica strain Z1978 culture was fermented and the microbialbiomass was harvested and dried, as described in the MATERIALS section,supra.

The dried and untreated biomass was then fed to a twin screw extruder.Specifically, a mixture of the biomass and 15% of diatomaceous earth(Celatom MN-4 or Celite 209, EP Minerals, LLC, Reno, Nev.) were premixedand then fed to a ZSK-40 mm MC twin screw extruder (Coperion Werner &Pfleiderer, Stuttgart, Germany) at a rate of 45.5 kg/hr. A water/sucrosesolution made of 26.5% sucrose was injected after the disruption zone ofthe extruder at a flow rate of 147 mL/min. The extruder was operated at280 rpm with a % torque range of 20-23. The resulting disrupted yeastpowder was cooled to 35° C. in a final water cooled barrel. The moistextruded powder was then fed into a LCI Dome Granulator Model No. TDG-80(LCI Corporation, Charlotte, N.C.) assembled with a multi-bore dome die1 mm diameter by 1 mm thick screen and set to 82 RPM. Extrudate wasformed at 455-600 kg/hr (as dried rate). The sample was dried in avibratory fluid bed dryer (FBP-75, Carman Industries, Inc.,Jeffersonville, Ind.) with a drying zone of 0.50 m² with 1150 standardcubic feet per minute [“scfm”] of air flow maintained at 100° C. and acooling zone of 0.24 m² operating with an air flow estimated at 500-600scfm at 18° C. Dried pellets, approximately 1 mm diameter×6 to 10 mm inlength, exited the dryer in the 25-30° C. range, having a final moisturecontent of 5-6% measured on an O'Haus moisture analyzer (Parsippany,N.J.).

Oil Extraction of the Extruded Yeast Biomass

The extruded yeast pellets were extracted using supercritical fluidphase CO₂ as the extraction solvent to produce non-concentratedextracted oil, using the 320 L stainless steel extraction vessel andconfiguration described in Example 22B. The oil extract was periodicallydrained from the separator and collected as product.

The extraction vessel was initially charged with approximately 150 kg ofthe extruded yeast pellets. The non-concentrated extracted oil was thenextracted from the pellets with supercritical fluid CO₂ at 5000 psig(345 bar), 55° C., and a solvent-to-feed ratio ranging from 40 to 50 kgCO₂ per kg of starting yeast pellets. Roughly 37.5 kg ofnon-concentrated extracted oil was collected from the separator vessel,to which was added about 1000 ppm each of two antioxidants, i.e. Covi-oxT70 (Cognis, Mississauga, Canada) and Dadex RM (Nealanders, Mississauga,Canada).

Distillation Under SPD Conditions

The non-concentrated extracted oil was degassed and then passed througha 6″ molecular still (POPE Scientific, Saukville, Wis.) using a feedrate of 12 kg/hr to remove residual water. The surface temperatures ofthe evaporator and condenser were set at 140° C. and 15° C.,respectively. The vacuum was maintained at 15 torr.

The dewatered extracted oil was passed through the molecular still at afeed rate of 12 kg/hr for a second time to remove undesiredlower-molecular weight compounds, such as ergosterol and free fattyacids in the distillate. The vacuum was lowered to 1 mtorr, and thesurface temperatures of the evaporator were maintained between 240° C.and 270° C. A triacylglycerol-containing fraction (i.e., thelipid-containing fraction or SPD-purified oil) was obtained, havingreduced sterols relative to the sterol content in the non-concentratedextracted oil. The non-concentrated SPD-purified oil was cooled to below40° C. before packaging.

Characterization of SPD-Purified Oil from Yarrowia lipolytica StrainZ1978

The fatty acid composition of the non-concentrated SPD-purified oil fromstrain Z1978 was analyzed, following transesterification, according tothe methodology of Example 27. The SPD-purified oil contained 56.1 EPA %TFAs and DHA was non-detectable (i.e. <0.05%), as shown below in Table34.

TABLE 34 Fatty Acid Composition Of Non-Concentrated Z1978 SPD-PurifiedOil Fatty acid Weight Percent Of Total Fatty Acids C18:2 (omega-6) 14.2C20:5 EPA 56.1 C22:6 DHA non-detectable (<0.05%) Other components 29.7

Example 32 Enrichment of Microbial Oil Via Fractional Distillation

This example demonstrates that an EPA concentrate comprising up to 74%EPA ethyl ester, measured as a weight percent of oil, and substantiallyfree of DHA could be obtained upon enrichment of the non-concentratedSPD-purified oil from Example 31 using a fractional distillation method.

Twenty-five (25) kg of the non-concentrated microbial oil from Example31 was added to a 50 L glass flask. 7.9 kg of absolute ethanol and 580 gof sodium ethoxide (21% in ethanol) were then added to the flask. Themixture was heated to reflux at ˜85° C. for a minimum of 30 min. Thereaction was monitored by a thin layer chromatography method, where adiluted sample of the oil was spotted onto a silica plate and separatedusing an acetic acid/hexane/ethyl ether solvent mixture. Spotsconsisting of unreacted TAGs were detected by iodine stain. Absent orbarely detectable spots were considered to represent completion of thereaction. After the reaction end point was reached, the mixture wascooled to below 50° C. and allowed to phase separate. Theglycerol-containing bottom layer was separated and discarded. The upperorganic layer was washed with 2.5 L of 5% citric acid, and the recoveredorganic layer was then washed with 5 L of 15% aqueous sodium sulfate.The aqueous phase was again discarded, and the ethyl ester phase wasdistilled with ethanol in a rotavap at ˜60° C. to remove residual water.Approximately 25 kg of oil in ethyl ester form was recovered.

The ethyl esters were then fed to a 4″ hybrid wiped-film andfractionation system (POPE Scientific, Saukville, Wis.) at a feed rateof 5 kg/hr to enrich EPA ethyl esters. The evaporator temperature wasset at approximately 275° C. under a vacuum of 0.47 torr. The headtemperature of the packed column was about 146° C. Thelower-molecular-weight ethyl esters, mainly C18s, were removed as alight fraction from the overhead. The extracted EPA ethyl esters wererecovered as a heavy fraction and underwent a second distillation,mainly for removing color and polymerized. The second distillation wasperformed in a 6″ molecular still (POPE Scientific, Saukville, Wis.) ata feed rate of 20 kg/hr. The evaporator was operated at about 205° C.with an internal condenser temperature setting of about 10° C. and avacuum of 0.01 torr. Approximately 7-10 wt % of the ethyl esters wasremoved, yielding a clear and light color EPA concentrate. The final EPAconcentrate contained 74% EPA ethyl esters, measured as a weight percentof oil, and substantially free of DHA.

One of skill in the art will appreciate that the EPA concentrate,comprising 74% EPA ethyl ester, measured as a weight percent of oil, andsubstantially free of DHA, could readily be converted to yield an EPAconcentrate in an alternate form (i.e., the EPA ethyl ester could beconverted to free fatty acids, triacylglycerols, methyl esters, andcombinations thereof), using means well known to those of skill in theart. Thus, for example, the 74% EPA ethyl ester could be re-esterifiedto triglycerides via glycerolysis, to result in an EPA concentrate, intriglyceride form, comprising at least 70 wt % of EPA, measured as a wt% of oil, and substantially free of DHA.

Example 33 EPA Concentrates are Substantially Free of EnvironmentalPollutants

This example demonstrates that both an EPA concentrate comprising atleast 70 wt % of EPA, measured as a wt % of oil, and substantially freeof DHA, and the microbial oil comprising 30-70 wt % of EPA, measured asa wt % of TFAs, and substantially free of DHA, are substantially free ofenvironmental pollutants.

A comparable sample of non-concentrated purified oil from Yarrowialipolytica strain Y8672 was prepared, as described in Example 27. Theconcentration, measured as mg/g World Health Organization InternationalToxicity Equivalent [“WHO TEQ”], of polychlorinated biphenyls [“PCBs”](CAS No. 1336-36-3), polychlorinated dibenzodioxins [“PCDDs”] andpolychlorinated dibenzofurans [“PCDFs”] in the non-concentratedextracted oil was determined according to EPA method 1668 Rev A.Extremely low or non-detectable levels of the environmental pollutantswere detected.

Based on the results above, it is assumed herein that the concentrationof PCBs, PCDDs, and PCDFs in the non-concentrated extracted oil ofExample 27 and the non-concentrated SPD-purified oil of Example 31 willalso contain extremely low or non-detectable levels of environmentalpollutants. Similarly, it is hypothesized herein that the EPA ethylester concentrates in Examples 28, 29, 30 and 32, enriched via ureaadduct formation, liquid chromatography, SFC and fractionaldistillation, respectively, should also contain extremely low ornon-detectable levels of environmental pollutants since they wereproduced from non-concentrated oils that are themselves substantiallyfree of environmental pollutants.

More specifically, Table 35 describes the expected TEQ levels of PCBs,PCDDs, and PCDFs within the EPA concentrates in Examples 28, 29, 30 and32. For comparison, the concentrations of the same compounds in apollutant-stripped marine oil described in U.S. Pat. No. 7,732,488 arealso included. It is noted that U.S. Pat. No. 7,732,488 provides specialprocessing methods to reduce these environmental pollutants toacceptable levels.

TABLE 35 Expected Environmental Pollutant Concentration (pg/g WHO TEQ)In EPA Concentrates EPA ethyl ester FIG. 2 from concentrates U.S. Pat.No. 7,732,488 Polychlorinated <0.1 0.17 Biphenyls (PCBs) Polychlorinated<0.1 0.26 Dibenzodioxins (PCDDs, dioxins) Polychlorinated non-detectable0.2 Dibenzofurans (<0.03) (PCDFs, furans)As shown above, the EPA ethyl ester concentrates in Examples 28, 29, 30and 32 will have lower levels of PCBs, PCDDs and PCDFs than thepollutant-stripped marine oil in U.S. Pat. No. 7,732,488. In fact, thepollutant level of PCDFs is expected to be below the detection limit ofthe analytical method used.

Example 34 Enrichment of Microbial Oil Via Fractional Distillation andLiquid Chromatography

This example demonstrates that an EPA concentrate comprising up to 97.4%EPA ethyl ester, measured as a weight percent of oil, and substantiallyfree of DHA, NDPA and HPA could be obtained upon enrichment of anon-concentrated purified oil using a combination of fractionaldistillation and liquid chromatography methods.

A lipid-containing fraction was obtained from Yarrowia lipolytica strainY9502 (supra, Example 31; see also U.S. Pat. Appl. Pub. No.2010-0317072-A1). Specifically, the strain was cultured, harvested,disrupted via extrusion and pelletization, and extracted usingsupercritical fluid phase CO₂ as described in Example 31. Thenon-concentrated extracted oil was then purified under SPD conditions(Example 31).

Characterization of SPD-Purified Oil from Yarrowia lipolytica StrainY9502

The fatty acid composition of the non-concentrated SPD-purified oil fromstrain Y9502 was analyzed according to the methodology of Example 27.The SPD-purified oil contained 54.7 EPA % TFAs and DHA, NDPA and HPAwere non-detectable (i.e., <0.05%), as shown below in Table 36.

TABLE 36 Fatty Acid Composition Of Non-Concentrated Y9502 SPD-PurifiedOil Fatty acid Weight Percent Of Total Fatty Acids C18:2 (omega-6) 15  C19:5 (omega-2) non-detectable (<0.05%) C20:5 EPA 54.7 C21:5 HPANon-detectable (<0.05%) C22:6 DHA non-detectable (<0.05%) Othercomponents 30.3Enrichment of SPD-Purified Oil from Yarrowia lipolytica Strain Y9502

The SPD-purified oil was transesterified to ethyl esters using a similarmethod as described in Example 29 and further subjected to fractionaldistillation as described in Example 31. The fractionally distilled EPAconcentrate contained 71.9% EPA ethyl esters, measured as a weightpercent of oil, and was substantially free of DHA, NDPA and HPA (see thecolumn titled “Fractionally Distilled” below in Table 37).

The fractionally distilled ethyl esters were then enriched by Equateq(Isle of Lewis, Scotland) using their liquid chromatographicpurification technology. The enrichment of the fractionally distilledEPA concentrate via liquid chromotography yielded a final EPAconcentrate with up to 97.4% EPA ethyl ester, measured as a weightpercent of oil, and substantially free of DHA, NDPA and HPA (see thecolumn titled “Liquid Chromotography Enriched” below in Table 37).

TABLE 37 EPA Ethyl Ester Concentrate With A Liquid ChromotographyEnrichment Method Weight Percent Of Oil Fatty acid Liquid Chromotographyethyl esters Fractionally Distilled Enriched C18:2 (omega-6)  0.8  0.05C19:5 NDPA Non-detectable (<0.05%) Non-detectable (<0.05%) (omega-2)C20:5 EPA 71.9 97.4 C21:5 HPA Non-detectable (<0.05%) Non-detectable(<0.05%) C22:6 DHA Non-detectable (<0.05%) Non-detectable (<0.05%) Othercomponents 27.3  2.1

One of skill in the art will appreciate that the EPA concentrate,comprising 97.4% EPA ethyl ester, measured as a weight percent of oil,and substantially free of DHA, NPDA and HPA, could readily be convertedto yield an EPA concentrate in an alternate form (i.e., the EPA ethylester could be converted to free fatty acids, triacylglycerols, methylesters, and combinations thereof), using means well known to those ofskill in the art. Thus, for example, the 97.4% EPA ethyl ester could bere-esterified to triglycerides via glycerolysis, to result in an EPAconcentrate, in triglyceride form, comprising at least 70 wt % of EPA,measured as a wt % of oil, and substantially free of DHA, NPDA and HPA.

Additionally, it is noted that EPA concentrates prepared according tothe methods of the invention herein from any microbial biomass ofrecombinant Yarrowia cells, engineered for the production of EPA, areexpected to be substantially free of DHA, NDPA and HPA. The resultsobtained above based on microbial oil obtained from Y. lipolytica strainY9502, wherein the final EPA concentrate is substantially free of DHA,NDPA and HPA, would be expected from EPA concentrates prepared frommicrobial oils obtained from Example 27 and Example 31. Since DHA, NDPAand HPA impurities are not present in the initial microbial oilcomprising 30 to 70 wt % of EPA, measured as a wt % of TFAs, obtainedfrom a Yarrowia that accumulates in excess of 25% of its dry cell weightas oil, the fatty acid impurities will also not be present in an EPAconcentrate produced therefrom.

1. A method comprising pelletizing a microbial biomass having a moisturelevel and comprising oil-containing microbes, wherein said pelletizingcomprises: (1) mixing the microbial biomass and at least one grindingagent capable of absorbing oil to provide a disrupted biomass mix; (2)blending the disrupted biomass mix with at least one binding agent toprovide a fixable mix capable of forming a solid pellet; and (3) formingsaid fixable mix into solid pellets to provide a pelletized microbialbiomass.
 2. The method of claim 1, wherein the oil-containing microbesare selected from the group consisting of yeast, algae, fungi, bacteria,euglenoids, stramenopiles, and oomycetes.
 3. The method of claim 1,wherein the oil-containing microbes comprise at least onepolyunsaturated fatty acid.
 4. The method of claim 1, wherein themoisture level of the microbial biomass is in the range of about 1 to 10weight percent.
 5. The method of claim 1, further comprising extractingthe pelletized microbial biomass to produce an extracted oil.
 6. Themethod of claim 1, wherein the mixing of step (1) is done in a twinscrew extruder comprising: (a) a total specific energy input (SEI) ofabout 0.04 to 0.4 KW/(kg/hr); (b) a compaction zone using bushingelements with progressively shorter pitch length; and (c) a compressionzone using flow restriction; wherein the compaction zone is prior to thecompression zone within the extruder.
 7. The method of claim 1, wherein:(a) said at least one grinding agent: (i) comprises a particle having aMoh hardness of 2.0 to 6.0 and an oil absorption coefficient of 0.8 orhigher as determined according to ASTM Method D1483-60; (ii) comprisessilica or silicate; or (iii) is present at about 1 to 20 weight percent,based on the summation of the weights of microbial biomass, grindingagent and binding agent in the solid pellets; and/or (b) said at leastone binding agent: (iv) comprises water or a carbohydrate selected fromthe group consisting of sucrose, lactose, fructose, glucose, and solublestarch; or (v) is present at about 0.5 to 10 weight percent, based onthe summation of the weights of microbial biomass, grinding agent andbinding agent in the solid pellets.
 8. The method of claim 1, wherein:steps (1) and (2) are performed in an extruder, are performedsimultaneously, or are performed simultaneously in an extruder; and/orstep (3) comprises a step selected from the group consisting of: (i)extruding said fixable mix through a die to form strands, (ii) dryingand breaking said strands, and (iii) a combination of step (i) and step(ii).
 9. The method of claim 1, wherein said solid pellets: have anaverage diameter of about 0.5 to about 1.5 mm and an average length ofabout 2.0 to about 8.0 mm, have a moisture level of about 0.1 to 5.0weight percent, or comprise about 70 to about 98.5 weight percent ofmicrobial biomass comprising oil-containing microbes, about 1 to about20 weight percent of at least one grinding agent capable of absorbingoil, and about 0.5 to 10 weight percent of at least one binding agent,based on the summation of the weights of microbial biomass, grindingagent and binding agent in the solid pellets.
 10. The method of claim 5,wherein said extracting is performed with an organic solvent and saidextracted oil is degummed and optionally bleached.
 11. The method ofclaim 5, wherein said extracting comprises: processing the pelletizedmicrobial biomass with a solvent comprising liquid carbon dioxide orsupercritical fluid carbon dioxide, wherein said pelletized microbialbiomass further comprises at least one polyunsaturated fatty acid, toobtain: (i) an extract comprising a lipid fraction substantially free ofphospholipids; and (ii) a residual biomass comprising phospholipids. 12.The method of claim 11, wherein said extracting further comprises:fractionating the extract comprising the lipid fraction substantiallyfree of phospholipids at least once to obtain an extracted oil having arefined lipid composition comprising at least one polyunsaturated fattyacid, wherein the refined lipid composition is enriched intriacylglycerols relative to the oil composition of pelletized microbialbiomass that is not processed with a solvent. 13-15. (canceled)
 16. Themethod of claim 2, wherein the oil-containing microbes are Yarrowiayeast.
 17. The method of claim 16, wherein the Yarrowia yeast arerecombinantly engineered for the production of a polyunsaturated fattyacid selected from the group consisting of: linoleic acid,gamma-linolenic acid, eicosadienoic acid, dihomo-gamma-linolenic acid,arachidonic acid, docosatetraenoic acid, omega-6 docosapentaenoic acid,alpha-linolenic acid, stearidonic acid, eicosatrienoic acid,eicosatetraenoic acid, eicosapentaenoic acid, omega-3 docosapentaenoicacid, and docosahexaenoic acid. 18-19. (canceled)
 20. The method ofclaim 11, wherein the residual biomass comprising phospholipids isfurther extracted to isolate the phospholipids.
 21. The method of claim17, wherein the recombinant Yarrowia yeast are engineered for productionof eicosapentaenoic acid.
 22. The method of claim 21, wherein at least20% of the dry cell weight of said Yarrowia yeast is eicosapentaenoicacid.